Compositions including triciribine and taxanes and methods of use thereof

ABSTRACT

This application relates to combination therapies including triciribine and related compounds and taxanes and compositions with reduced toxicity for the treatment and prevention of tumors, cancer, and other disorders associated with abnormal cell proliferation.

This application is a continuation of U.S. application Ser. No.14/091,073, filed Nov. 26, 2013, which is a continuation of U.S.application Ser. No. 13/452,478, filed Apr. 20, 2012, which is acontinuation of U.S. application Ser. No. 12/118,828, filed May 12,2008, which is abandoned, which is a continuation-in-part of U.S.application Ser. No. 11/096,082, filed Mar. 25, 2005, and issued as U.S.Pat. No. 8,435,959 on May 7, 2013, which claims the benefit of U.S.provisional patent application No. 60/557,599 filed Mar. 29, 2004, whichis incorporated herein by reference.

1. FIELD OF THE INVENTION

This application relates to combination therapies including triciribinecompounds and one or more taxanes and compositions including suchcombinations with reduced toxicity for the treatment and prevention oftumors, cancer, and other disorders associated with abnormal cellproliferation.

2. BACKGROUND OF THE INVENTION

Cancer is an abnormal growth of cells. Cancer cells rapidly reproducedespite restriction of space, nutrients shared by other cells, orsignals sent from the body to stop reproduction. Cancer cells are oftenshaped differently from healthy cells, do not function properly, and canspread into many areas of the body. Abnormal growths of tissue, calledtumors, are clusters of cells that are capable of growing and dividinguncontrollably. Tumors can be benign (noncancerous) or malignant(cancerous). Benign tumors tend to grow slowly and do not spread.Malignant tumors can grow rapidly, invade and destroy nearby normaltissues, and spread throughout the body.

Cancers are classified according to the kind of fluid or tissue fromwhich they originate, or according to the location in the body wherethey first developed. In addition, some cancers are of mixed types.Cancers can be grouped into five broad categories, carcinomas, sarcomas,lymphomas, leukemias, and myelomas, which indicate the tissue and bloodclassifications of the cancer. Carcinomas are cancers found in bodytissue known as epithelial tissue that covers or lines surfaces oforgans, glands, or body structures. For example, a cancer of the liningof the stomach is called a carcinoma. Many carcinomas affect organs orglands that are involved with secretion, such as breasts that producemilk. Carcinomas account for approximately eighty to ninety percent ofall cancer cases. Sarcomas are malignant tumors growing from connectivetissues, such as cartilage, fat, muscle, tendons, and bones. The mostcommon sarcoma, a tumor on the bone, usually occurs in young adults.Examples of sarcoma include osteosarcoma (bone) and chondrosarcoma(cartilage). Lymphoma refers to a cancer that originates in the nodes orglands of the lymphatic system, whose job it is to produce white bloodcells and clean body fluids, or in organs such as the brain and breast.Lymphomas are classified into two categories: Hodgkin's lymphoma andnon-Hodgkin's lymphoma. Leukemia, also known as blood cancer, is acancer of the bone marrow that keeps the marrow from producing normalred and white blood cells and platelets. White blood cells are needed toresist infection. Red blood cells are needed to prevent anemia.Platelets keep the body from easily bruising and bleeding. Examples ofleukemia include acute myelogenous leukemia, chronic myelogenousleukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia.The terms myelogenous and lymphocytic indicate the type of cells thatare involved. Finally, myelomas grow in the plasma cells of bone marrow.In some cases, the myeloma cells collect in one bone and form a singletumor, called a plasmacytoma. However, in other cases, the myeloma cellscollect in many bones, forming many bone tumors. This is called multiplemyeloma.

Tumor induction and progression are often the result of accumulatedchanges in the tumor-cell genome. Such changes can include inactivationof cell growth inhibiting genes, or tumor suppressor genes, as well asactivation of cell growth promoting genes, or oncogenes. Hundreds ofactivated cellular oncogenes have been identified to date in animalmodels, however, only a small minority of these genes have proven to berelevant to human cancers (Weinberg et al 1989 Oncogenes and theMolecular Origins of Cancer Cold Spring Harbor, N.Y., Stanbridge andNowell 1990 Cell 63 867-874, Godwin et al 1992 Oncogenes andantioncogenes in gynecological malignancies. In WJ Hoskins, C A Perezand RC Young (eds), Gynecological oncology: principles and practice, pp87-116, Lippincott, Philadelphia). The activation of oncogenes in humancancers can result from factors such as increased gene copy number orstructural changes. These factors can cause numerous cellular effects,for example, they can result in overexpression of a gene product.Several oncogenes involved in human cancer can be activated through geneoverexpression.

It has become apparent that the successive genetic aberrations acquiredby cancer cells result in defects in regulatory signal transductioncircuits that govern normal cell proliferation, differentiation andprogrammed cell death (Hanahan, D. and R. A. Weinberg, Cell, 2000.100(1): p. 57-700). This in turn results in fundamental defects in cellphysiology which dictate malignancy. These defects include: a) selfsufficiency in growth signals (i.e. overexpression of growth factorreceptor tyrosine kinases such as EGFR and aberrant activation ofdownstream signal transduction pathways such as Ras/Raf/Mek/Erk 1/2 andRas/PI3K/Akt), b) resistance to anti-growth signals (i.e. lowerexpression of TGFβ and its receptor), c) evading apoptosis (i.e. loss ofproapoptotic p53; overexpression of pro-survival Bcl-2; hyperactivationof survival pathways such as those mediated by PI3K/Akt), d) sustainedangiogenesis (i.e. high levels of secretion of VEGF) and f) tissueinvasion and metastasis (i.e. extracellular proteases and prometastaticintegrins) (Hanahan, D. and R. A. Weinberg, Cell, 2000. 100(1): p.57-700).

Receptor tyrosine kinases such as EGFR, ErbB2, VEGFR and insulin-likegrowth factor I receptor (IGF-1R) are intimately involved in thedevelopment of many human cancers including colorectal pancreatic,breast and ovarian cancers (Khaleghpour, K., et al. Carcinogenesis,2004. 25(2): p. 241-8; Sekharam, M., et al., Cancer Res, 2003. 63(22):p. 7708-16). Binding of ligands such as EGF, VEGF and IGF-1 to theirreceptors promotes stimulation of the intrinsic tyrosine kinaseactivity, autophosphorylation of specific tyrosines in the cytoplasmicdomain of the receptors and recruitment of signaling proteins thattrigger a variety of complex signal transduction pathways (Olayioye, M.A., et al., Embo J, 2000. 19(13): p. 3159-67, Porter, A. C. and R. R.Vaillancourt, Oncogene, 1998. 17(11 Reviews): p. 1343-52).. This in turnleads to the activation of many tumor survival and oncogenic pathwayssuch as the Ras/Raf/Mek/Erk 1/2, JAK/STAT3 and PI3K/Akt pathways.Although all three pathways have been implicated in colon, pancreatic,breast and ovarian oncogenesis, those that are mediated by Akt have beenshown to be critical in many steps of malignant transformation includingcell proliferation, anti-apoptosis/survival, invasion and metastasis andangiogenesis (Datta, S. R. et al. Genes Dev, 1999. 13(22): p. 2905-27).

Akt is a serine/threonine protein kinase (also known as PK_(B)), whichhas 3 family members Akt1, Akt2 and Akt3. Stimulation of cells withgrowth or survival factors results in recruitment to the receptors ofthe lipid kinase phosphoinositide-3-OH-kinase (PI3K) whichphosphorylates phosphoinositol-4,5-biphosphate (PIP₂) to PIP₃ whichrecruits Akt to the plasma membrane where it can be activated byphosphorylation on Thr308 and Ser473 (Akt1), Thr308 and Ser474 (Akt2)and Thr308 and Ser472 (Akt3) (Datta, S. R. et al. Genes Dev, 1999.13(22): p. 2905-27). Thus, PI3K activates Akt by phosphorylating PIP2and converting to PIP3. The phosphatase PTEN dephophorylates PIP3 toPIP2 and hence prevents the activation of Akt.

The majority of human cancers contain hyperactivated Akt (Datta, S. R.et al. Genes Dev, 1999. 13(22): p. 2905-27, Bellacosa, A., et al., Int JCancer, 1995. 64(4): p. 280-5; Sun, M., et al., Am J Pathol, 2001.159(2): p. 431-7). In particular, Akt is overexpressed and/orhyperactivated in 57%, 32%, 27% and 36% of human colorectal, pancreatic,breast and ovarian cancers, respectively (Roy, H. K., et al.Carcinogenesis, 2002. 23(1): p. 201-5. Altomare, D. A., et al., J CellBiochem, 2003. 88(1): p. 470-6, Sun, M., et al., Cancer Res, 2001.61(16): p. 5985-91, Stal, O., et al. Breast Cancer Res, 2003. 5(2): p.R37-44, Cheng, J. Q., et al., Proc Natl Acad Sci USA, 1992. 89(19): p.9267-71, Yuan, Z. Q., et al., Oncogene, 2000. 19(19): p. 2324-30).Hyperactivation of Akt is due to amplification and/or overexpression ofAkt itself as well as genetic alterations upstream of Akt includingoverexpression of receptor tyrosine kinases and/or their ligands(Khaleghpour, K., et al. Carcinogenesis, 2004. 25(2): p. 241-8;Sekharam, M., et al., Cancer Res, 2003. 63(22): p. 7708-16, Cohen, B.D., et al., Biochem Soc Symp, 1998. 63: p. 199-210, Muller, W. J., etal. Biochem Soc Symp, 1998. 63: p. 149-57, Miller, W. E., et al. JVirol, 1995. 69(7): p. 4390-8, Slamon, D. J., et al., Science, 1987.235(4785): p. 177-82, Andrulis, I. L., et al., J Clin Oncol, 1998.16(4): p. 1340-9) and deletion of the phosphatase PTEN. Proof-of-conceptof the involvement of Akt in oncogenesis has been demonstratedpreclinically by showing that ectopic expression of Akt inducesmalignant transformation and promotes cell survival (Sun, M., et al. AmJ Pathol, 2001. 159(2): p. 431-7, Cheng, J. Q., et al., Oncogene, 1997.14(23): p. 2793-801) and that disruption of Akt pathways inhibits cellgrowth and induces apoptosis (Jetzt, A., et al. Cancer Res, 2003.63(20): p. 6697-706).

Current treatments of cancer and related diseases have limitedeffectiveness and numerous serious unintended side effects. Despitedemonstrated clinical efficacy of many anti-cancer drugs, severesystemic toxicity often halts the clinical development of promisingchemotherapeutic agents. Further, overexpression of receptor tyrosinekinases such as EGFR and their ligands such as IGF-1 and Aktoverexpression and/or loss of PTEN (all of which result inhyperactivation of Akt) are associated with poor prognosis, resistanceto chemotherapy and shortened survival time of cancer patients. Currentresearch strategies emphasize the search for effective therapeutic modeswith less risk.

3. SUMMARY OF THE INVENTION

The present invention provides novel therapeutic regimens oftriciribine, triciribine phosphate and related compounds in combinationwith one or more taxanes to treat tumors or cancer in a subject whilelimiting systemic toxicity. The invention is based on the discovery thattumors or cancers, which overexpress Akt kinase are particularlysensitive to the cytotoxic effects of TCN and related compounds and asynergistic affect arises with a combination of at least one taxane. Theinventors have determined, contrary to the prior knowledge andexperience, how to successfully use a combination of triciribine andrelated compounds and at least one taxane to treat tumors and cancer byone or a combination of (i) administering triciribine and a taxane topatients who exhibit enhanced sensitivity to the triciribine; (ii) useof a described dosage level that minimizes the toxicity of the drugs butyet still exhibits efficacy; or (iii) use of a described dosage regimenthat minimizes the toxicity of the drugs.

In one aspect of the present invention, the invention encompasses acomposition including:

(i) a compound of the formula I:

wherein each R2′, R3′ and R5′ are independently hydrogen, optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as for example as described in the definition of an arylgiven herein; optionally substituted arylsulfonyl; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group that, in vivo,provides a compound wherein R2′, R3′ or R5′ is independently H or mono-,di- or tri-phosphate; wherein R^(x) and R^(y) are independentlyhydrogen, optionally substituted phosphate; acyl (including lower acyl);amide, alkyl (including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl;

(ii) a compound of formula II:

wherein

R¹⁰ and R¹¹ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, aryl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl; and

(iii) a pharmaceutically acceptable carrier.

In another embodiment, the invention encompasses a method of treating atumor or cancer in a mammal including administering to the mammal aneffective amount of a composition including:

(i) a compound of formula I:

wherein each R2′, R3′ and R5′ are independently hydrogen, optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as for example as described in the definition of an arylgiven herein; optionally substituted arylsulfonyl; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group that, in vivo,provides a compound wherein R2′, R3′ or R5′ is independently H or mono-,di- or tri-phosphate; wherein R^(x) and R^(y) are independentlyhydrogen, optionally substituted phosphate; acyl (including lower acyl);amide, alkyl (including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl; and

(ii) a compound of formula II:

wherein

R¹⁰ and R¹¹ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In one particular embodiment, the compound of formula II has thefollowing structure:

Methods are useful to treat tumors and cancers that are particularlysusceptible to the toxic effects of TCN, TCN-P and/or related compounds.In another embodiment, methods are provided for treating a tumor in amammal, particularly a human that includes (i) obtaining a biologicalsample from the tumor; (ii) determining whether the tumor overexpressesan Akt kinase, and (iii) treating the tumor that overexpresses Aktkinase with triciribine, triciribine phosphate or a related compound incombination with one or more taxanes, as described herein. In anotherembodiment, the level of Akt kinase expression can be determined byassaying the tumor or cancer for the presence of a phosphorylated Aktkinase, for example, by using an antibody that can detect thephosphorylated form. In another embodiment, the level of Akt expressioncan be determined by assaying a tumor or cancer cells obtained from asubject and comparing the levels to a control tissue. In certainembodiments, the Akt can be overexpressed at least 2, 2.5, 3 or 5 foldin the cancer sample compared to the control. In certain embodiments,the overexpressed Akt kinase can be a hyperactivated and phosphorylatedAkt kinase.

In another aspect of the invention, dosing regimens are provided thatlimit the toxic side effects of TCN and related compounds. In anotherembodiment, such dosing regimens minimize or eliminate toxic sideeffects, including, but not limited to, hepatoxicity, thrombocytopenia,hyperglycemia, vomiting, hypocalcemia, anemia, hypoalbunemia,myelosuppression, hypertriglyceridemia, hyperamylasemia, diarrhea,stomachitis and/or fever. In another embodiment, the administration ofTCN, TCN-P or related compounds provides at least a partial, such as atleast 15, 20 or 30%, or complete response in vivo in at least 15, 20, or25% of the subjects.

In another embodiment, a method is provided to treat a subject which hasbeen diagnosed with a tumor by administering to the subject an effectiveamount of TCN, TCN-P or a related compound and one or more taxanesaccording to a dosing schedule that includes administering the drugapproximately one time per week for approximately three weeks followedby a one week period wherein the drug is not administered. In anotherembodiment, methods are provided to treat tumor or cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P or a related compound and one or more taxanes each one timeper week. In another embodiment, the triciribine compound and one ormore taxanes can be administered as a single bolus dose over a shortperiod of time, for example, about 5, 10 or 15 minutes. In furtherembodiments, dosing schedules are provided in which the triciribinecompound and one or more taxanes are administered via continuousinfusion for at least 24, 48, 72, 96, or 120 hours. In certainembodiments, the continuous administration can be repeated at least oncea week, once every two weeks and/or once a month. In other embodiments,the triciribine compound and one or more taxanes can be administered atleast once every three weeks. In further embodiments, the compounds canbe administered at least once a day for at least 2, 3, 4 or 5 days.

In further embodiments, the triciribine compound and one or more taxanesas disclosed herein can be administered to patients in an amount that iseffective in causing tumor regression. The administration thetriciribine compound and one or more taxanes can provide at least apartial, such as at least 15, 20 or 30%, or complete response in vivo inat least 15-20% of the subjects. In certain embodiments, at least about2, 5, 10, 15, 20, 30 or 50 mg/m² of the triciribine compound and atleast about 0.1, 1, 2, 5, 10, 15, 20, 30 or 50 mg of one or more taxanesdisclosed herein can be administered to a subject. The administration ofthe triciribine compound and one or more taxanes can be conductedaccording to any of the therapeutic regimens disclosed herein. Inparticular embodiments, the dosing regimen can include administeringless than 20 mg/m² of the triciribine compound and one or more taxanes.In one embodiment, less than 10 mg/m² of the triciribine compound andone or more taxanes can be administered once a week. In furtherembodiments, dosages of or less than 2 mg/m², 5 mg/m², 10 mg/m², and/or15 mg/m² of the triciribine compound and at least about 0.1, 1, 2, 5,10, 15, 20, 30 or 50 mg one or more taxanes can be administered to asubject. In another embodiment, less than 10 mg/m² of triciribine can beadministered to a subject via continuous infusion for at least fivedays. In particular embodiments, the triciribine compound and one ormore taxanes as disclosed herein can be used for the treatment ofpancreatic, prostate, colo-rectal and/or ovarian cancer.

In another embodiment, the triciribine compound and one or more taxanesand/or therapeutic regimens of the present invention can be used toprevent and/or treat a carcinoma, sarcoma, lymphoma, leukemia, and/ormyeloma. In other embodiments of the invention, the triciribine compoundand one or more taxanes can be used to treat solid tumors. In stillfurther embodiments, the triciribine compound and one or more taxanesand compositions disclosed herein can be used for the treatment of atumor or cancer, such as, but not limited to cancer of the followingorgans or tissues: breast, prostate, bone, lung, colon, including, butnot limited to colorectal, urinary, bladder, non-Hodgkin lymphoma,melanoma, kidney, renal, pancreas, pharnx, thyroid, stomach, brain,and/or ovaries. In a particular embodiment, the triciribine compound andone or more taxanes can be used for the treatment of pancreatic, breast,colorectal and/or ovarian cancer. In further embodiments of the presentinvention, the triciribine compound and one or more taxanes disclosedherein can be used in the treatment of angiogenesis-related diseases. Incertain embodiments, methods are provided to treat leukemia viacontinuous infusion of, the triciribine compound and one or more taxanesvia continuous infusion for at least 24, 48, 72 or 96 hours. In otherembodiments, the continuous infusion can be repeated, for example, atleast once every two, three or four weeks.

In a particular embodiment, there is provided a method for the treatmentof tumors, cancer, and others disorders associated with an abnormal cellproliferation in a host, the method including administering to the hostan effect amount of the triciribine compound and one or more taxanesoptionally in combination with a pharmaceutically acceptable carrier.

In one aspect, the triciribine compound and one or more taxanes andcompositions can be administered in combination and can form part of thesame composition, or be provided as a separate composition foradministration at the same time or a different time.

In other embodiments, the triciribine compound and one or more taxanesas disclosed herein can be used to treat tumors or cancers resistant toone or more drugs, including the embodiments of tumors or cancers anddrugs disclosed herein. In one embodiment, the triciribine compound andone or more taxanes as disclosed herein is administered in an effectiveamount for the treatment of a patient with a drug resistant tumor orcancer, for example, multidrug resistant tumors or cancer including, butnot limited to, those resistant to taxol alone, rapamycin, tamoxifen,cisplatin, and/or gefitinib (iressa).

In certain embodiments, a method is provided including administering toa host in need thereof an effective amount of a triciribine compound andone or more taxanes disclosed herein, or pharmaceutical compositionincluding a triciribine compound and one or more taxanes, in aneffective amount for the treatment of tumors, cancer, and othersdisorders associated with an abnormal cell proliferation in a host.

In another embodiment, a method for the treatment of a tumor or canceris provided including an effective amount of a triciribine compound andone or more taxanes disclosed herein, or a salt, isomer, prodrug orester thereof, to an individual in need thereof, wherein the cancer isfor example, carcinoma, sarcoma, lymphoma, leukemia, or myeloma. Thecompound, or salt, isomer, prodrug or ester thereof, is optionallyprovided in a pharmaceutically acceptable composition including theappropriate carriers, such as water, which is formulated for the desiredroute of administration to an individual in need thereof. Optionally thecompound is administered in combination or alternation with at least oneadditional therapeutic agent for the treatment of tumors or cancer.

Also within the scope of the invention is the use of a triciribinecompound and one or more taxanes disclosed herein or a salt, prodrug orester thereof in the treatment of a tumor or cancer, optionally in apharmaceutically acceptable carrier; and the use of a triciribinecompound and one or more taxanes disclosed herein or a salt, prodrug orester thereof in the manufacture of a medicament for the treatment ofcancer or tumor, optionally in a pharmaceutically acceptable carrier.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D demonstrates the identification of API-2 (triciribine) as acandidate of Akt inhibitor from the NCI Diversity Set. A illustrates thechemical structure of API-2 (triciribine). B demonstrates that API-2inhibits phosphorylation levels of AKT2 in AKT2-transformed NIH3T3cells. Wile type AKT2-transformed NIH3T3 cells were treated with API-2(1 M) for indicated times and subjected to immunoblotting analysis withanti-phospho-Akt-T308 and -S473 antibodies (top and middle panels). Thebottom panel shows expression of total AKT2. In C, it is shown thatAPI-2 inhibits three isoforms of Akt. HEK293 cells were transfected withHA-Akt1, -AKT2 and -AKT3 and treated with API-2 (1 uM) or wortmannin (15uM) prior to EGF stimulation, the cells were lysed andimmunoprecipitated with anti-HA antibody. The immunoprecipitates weresubjected to in vitro kinase assay (top) and immunoblotting analysiswith anti-phospho-Akt-T308 (bottom) antibody. Middle panel showsexpression of transfected Akt1, AKT2 and AKT3. D illustrates that API-2did not inhibit Akt in vitro. In vitro kinase assay of constitutivelyactive AKT2 recombinant protein in a kinase buffer containing 1 uM API-2(lane 3).

FIGS. 2A-2F demonstrates that API-2 does not inhibit PI3K, PDK1 and theclosely related members of AGC kinase family. A demonstrates an in vitroPI3K kinase assay. HEK293 cells were serum-starved and treated withAPI-2 (1 uM) or Wortmannin (15 uM) for 30 minutes prior to EGFstimulation. Cells were lysed and immunoprecipitated with anti-p110aantibody. The immunoprecipitates were subjected to in vitro kinase assayusing PI-4-P as substrate. B illustrates the effect of API-2 on in vitroPDK1 activation (top panel), closed circles show inhibition by API-2.Open circles show inhibition by the positive control staurosporine,which is a potent PDK1 inhibitor (IC50=5 nM). Bottom panels areimmunoblotting analysis of HEK293 cells that were transfected withMyc-PDK1 and treated with wortmannin or API-2 prior to EGF stimulation.The immunoblots were detected with indicated antibodies. C illustratesan immunoblot analysis of phosphorylation levels of PKCα withanti-phospho-PKCα-T638 (top) and total PKCα (bottom) antibodiesfollowing treatment with API-2 or a nonselective PKC inhibitorRo31-8220. D shows an in vitro SGK kinase assay. HEK293 cells weretransfected with HA-SGK and treated with API-2 or wortmannin prior toEGF stimulation. In vitro kinase was performed with HA-SGKimmunoprecipitates using MBP as substrate (top). Bottom panel shows theexpression of transfected HA-SGK. E illustrates the results of a PKAkinase assay. Immuno-purified PKA was incubated in ADB buffer (UpstateBiotechnology Inc) containing indicated inhibitors (API-2 or PKAI) andsubstrate Kemptide. The kinase activity was quantified. In F, a westernblot is shown. OVCAR3 cells were treated with API-2 for indicated times.Cell lysates were immunoblotted with indicated anti-phospho-antibodies(panels 1-4) and anti-actin antibody (bottom).

FIGS. 3A-3C7 demonstrates that API-2 inhibits Akt activity and cellgrowth and induces apoptosis in human cancer cells with elevated Akt. Ais a western blot, following treatment with API-2, phosphorylationlevels of Akt were detected with anti-phospho-Akt-T308 antibody inindicated human cancer cell lines. The blots were reprobed withanti-total Akt antibody (bottom panels). In B, a cell proliferationassay is shown. Cell lines as indicated in the figure were treated withdifferent doses of API-2 for 24 h and 48 h and then analyzed withCellTiter 96 Cell Proliferation Assay kit (Promega). C provides anapoptosis analysis. Cells were treated with API-2 and stained withannexin V and PI and analyzed by FACScan.

FIGS. 4A-4E shows that API-2 inhibits downstream targets of Akt andexhibits anti-tumor activity in cancer cell lines with elevated Akt inmouse xenograft. In A, it is demonstrated that API-2 inhibits Aktphosphorylation of tuberin, Bad, AFX and GSK-3β. Following treatmentwith API-2, OVAR3 cells were lysed and immunoblotted with indicatedantibodies. B shows that API-2 inhibits tumor growth. Tumor cells weresubcutaneously injected into nude mice with low level of Akt cells onleft side and elevated level of Akt cells on right side. When the tumorsreached an average size of about 100-150 mm³, animals were treated witheither vehicle or 1 mg/kg/day API-2. Each measurement represents anaverage of 10 tumors. C illustrates a representation of the mice withOVCAR3 (right) and OVCAR5 (left) xenograft treated with API-2 or vehicle(control). D shows examples of tumor size (bottom) and weight (top) atthe end of experiment. In E, immunoblot analysis of tumor lysates wasperformed with anti-phospho-Akt-S473 (top) and anti-AKT2 (bottom)antibodies in OVCAR-3-derived tumors that were treated (T3 and T4) anduntreated (T1 and T2) with API-2.

FIG. 5 shows that API-2 (triciribine) inhibits Akt kinase activity invitro. In vitro kinase assay was performed with recombinant of PDK1 andAkt in a kinase buffer containing phosphatidylinositol-3,4,5-P3 (PIP3),API-2 and histone H2B as substrate. After incubation of 30 min, thereactions were separated by SDS-PAGE and exposed in a film.

FIGS. 6A-6D provides the mRNA (SEQ ID NO: 1) and amino acid sequence(SEQ ID NO: 2) of human Akt1, restriction enzyme sites are also noted.

FIGS. 7A-7D provides the mRNA (SEQ ID NO: 3) and amino acid sequence(SEQ ID NO:4) of human Akt2 restriction enzyme sites are also noted.

FIGS. 8A-8D provides the mRNA (SEQ ID NO. 5) and amino acid sequence(SEQ ID NO. 6) of human Akt3 restriction enzyme sites are also noted.

FIG. 9 shows the synergistic effect of a combination of triciribine andTaxol on different cancer cell lines.

5. DETAILED DESCRIPTION OF THE INVENTION

The inventors have determined, contrary to the prior art and experience,how to successfully use triciribine compounds in combination with one ormore taxanes to treat tumors and cancer by one or a combination of (i)administering triciribine and one or more taxanes only to patients whichaccording to a diagnostic test described below, exhibit enhancedsensitivity to the triciribine compound and/or the taxane; (ii) using adescribed dosage level that minimizes the toxicity of the triciribinecompound and/or the taxane but yet still exhibits efficacy; or (iii)using a described dosage regimen that minimizes the toxicity of thetriciribine compound and/or the taxane.

5.1. Definitions

As used herein, the term “compounds of the invention” refers tocompounds of formula I, compounds of formula II, and combinationsthereof.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth, i.e., proliferative disorders. Examples ofsuch proliferative disorders include cancers such as carcinoma,lymphoma, blastoma, sarcoma, and leukemia, as well as other cancersdisclosed herein. More particular examples of such cancers includebreast cancer, prostate cancer, colon cancer, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, cervical cancer, ovarian cancer, livercancer, e.g., hepatic carcinoma, bladder cancer, colorectal cancer,endometrial carcinoma, kidney cancer, and thyroid cancer.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CNS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); pancreatic cancer; retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; sarcoma; skin cancer;stomach cancer; testicular cancer; uterine cancer; cancer of the urinarysystem, as well as other carcinomas and sarcomas.

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid mass tumor. The solid tumor mass, if present,may be a primary tumor mass. A primary tumor mass refers to a growth ofcancer cells in a tissue resulting from the transformation of a normalcell of that tissue. In most cases, the primary tumor mass is identifiedby the presence of a cyst, which can be found through visual orpalpation methods, or by irregularity in shape, texture or weight of thetissue. However, some primary tumors are not palpable and can bedetected only through medical imaging techniques such as X-rays (e.g.,mammography), or by needle aspirations. The use of these lattertechniques is more common in early detection. Molecular and phenotypicanalysis of cancer cells within a tissue will usually confirm if thecancer is endogenous to the tissue or if the lesion is due to metastasisfrom another site.

The term alkyl, as used herein, unless otherwise specified, includes asaturated straight, branched, or cyclic, primary, secondary, or tertiaryhydrocarbon of for example C₁ to C₂₄, and specifically includes methyl,trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,cyclohexyl, cyclohexylmethyl, β-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The alkyl is optionally substituted, e.g., with oneor more substituents such as halo (F, Cl, Br or I), (e.g. CF₃,2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃ or CF₂CF₃), hydroxyl (e.g. CH₂OH), amino(e.g. CH₂NH₂, CH₂NHCH₃ or CH₂N(CH₃)₂), alkylamino, arylamino, alkoxy,aryloxy, nitro, azido (e.g. CH₂N₃), cyano (e.g. CH₂CN), sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term lower alkyl, as used herein, and unless otherwise specified,refers to a C₁ to C₄ saturated straight, branched, or if appropriate, acyclic (for example, cyclopropyl) alkyl group, including bothsubstituted and unsubstituted forms.

The term alkylamino or arylamino includes an amino group that has one ortwo alkyl or aryl substituents, respectively.

The term amino acid includes naturally occurring and synthetic α, β, γor δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration. Alternatively, the amino acid can be a derivativeof alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl,argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl,β-prolinyl, 0-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl,β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl,β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl orβ-histidinyl. When the term amino acid is used, it is considered to be aspecific and independent disclosure of each of the esters of a naturalor synthetic amino acid, including but not limited to α, β, γ or δglycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginineand histidine in the D and L-configurations.

The term “protected” as used herein and unless otherwise definedincludes a group that is added to an oxygen, nitrogen, sulfur orphosphorus atom to prevent its further reaction or for other purposes. Awide variety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis (see Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3^(rd) Ed., John Wiley & Sons, Inc., NewYork, N.Y., 1999).

The term aryl, as used herein, and unless otherwise specified, includesphenyl, biphenyl, or naphthyl, and preferably phenyl. The aryl group isoptionally substituted with one or more moieties such as halo, hydroxyl,amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, phosphonic acid, phosphate, or phosphonate, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as taught in Greene, et al., Protective Groups inOrganic Synthesis, John Wiley and Sons, 3^(rd) Ed., 1999.

The term alkaryl or alkylaryl includes an alkyl group with an arylsubstituent. The term aralkyl or arylalkyl includes an aryl group withan alkyl substituent.

The term halo, as used herein, includes chloro, bromo, iodo, and fluoro.

The term acyl includes a carboxylic acid ester in which the non-carbonylmoiety of the ester group is selected from straight, branched, or cyclicalkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkylincluding benzyl, aryloxyalkyl such as phenoxymethyl, aryl includingphenyl optionally substituted with halogen, C₁ to C₄ alkyl or C₁ to C₄alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxytrityl, substituted benzyl, trialkylsilyl (e.g.dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the estersoptimally include a phenyl group. The term “lower acyl” refers to anacyl group in which the non-carbonyl moiety is lower alkyl.

As used herein, the term “substantially free of” or “substantially inthe absence of” with respect to enantiomeric purity, refers to acomposition that includes at least 85% or 90% by weight, preferably 95%to 98% by weight, and even more preferably 99% to 100% by weight, of thedesignated enantiomer. In a preferred embodiment, in the methods andcompounds of this invention, the compounds are substantially free ofother enantiomers.

Similarly, the term “isolated” refers to a compound composition thatincludes at least 85% or 90% by weight, preferably 95% to 98% by weight,and even more preferably 99% to 100% by weight, of the compound, theremainder including other chemical species or enantiomers.

The term “independently” is used herein to indicate that the variable,which is independently applied, varies independently from application toapplication. Thus, in a compound such as R″XYR″, wherein R″ is“independently carbon or nitrogen,” both R″ can be carbon, both R″ canbe nitrogen, or one R″ can be carbon and the other R″ nitrogen.

The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a compound, which, upon administration to a patient, providesthe compound. Pharmaceutically acceptable salts include those derivedfrom pharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart. Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on afunctional moiety of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated, dephosphorylated to produce theactive compound.

The term “pharmaceutically acceptable esters” as used herein, unlessotherwise specified, includes those esters of one or more compounds,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of hosts without undue toxicity, irritation,allergic response and the like, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

The term “subject” as used herein refers to an animal, preferably amammal, most preferably a human. Mammals can include non-human mammals,including, but not limited to, pigs, sheep, goats, cows (bovine), deer,mules, horses, monkeys and other non-human primates, dogs, cats, rats,mice, rabbits or any other known or disclosed herein.

The term “taxanes” includes compounds encompassed by formula II orformula III.

The term TCN, TCN-P and related compounds includes compounds encompassedby formula I.

5.2. Compounds of the Invention

The present invention provides for the use of TCN, TCN-P and relatedcompounds in combination with one or more taxanes for use in particulartherapeutic regimens for the treatment of proliferative disorders.

As used herein and unless otherwise indicated, the term “triciribinecompounds” and “triciribine and related compounds” refers to compoundshaving the following structures:

wherein each R2′, R3′ and R5′ are independently hydrogen, optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as for example as described in the definition of an arylgiven herein; optionally substituted arylsulfonyl; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group that, in vivo,provides a compound wherein R2′, R3′ or R5′ is independently H or mono-,di- or tri-phosphate; wherein R^(x) and R^(y) are independentlyhydrogen, optionally substituted phosphate; acyl (including lower acyl);amide, alkyl (including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In one embodiment, R2′ and R3′ are hydrogen. In another embodiment, R2′and R5′ are hydrogen. In yet another embodiment, R2′, R3′ and R5′ arehydrogen. In yet another embodiment, R2′, R3′, R5′, R1 and R2 arehydrogen.

In another embodiment, the triciribine compound has the followingstructure:

wherein R₃ is H, optionally substituted straight chained, branched orcyclic alkyl (including lower alkyl), alkenyl, or alkynyl, NH₂, NHR⁴,N(R⁴)₂, aryl, alkoxyalkyl, aryloxyalkyl, or substituted aryl; and

Each R⁴ independently is H, acyl including lower acyl, alkyl includinglower alkyl such as but not limited to methyl, ethyl, propyl andcyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxyalkyl,hydroxyalkyl, or aryl. In a subembodiment, R₃ is a straight chainedC1-11 alkyl, iso-propyl, t-butyl, or phenyl.

In one embodiment, the triciribine compounds provided herein have thefollowing structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

wherein R₆ is H, alkyl, (including lower alkyl) alkenyl, alkynyl,alkoxyalkyl, hydroxyalkyl, arylalkyl, cycloalkyl, NH₂, NHR⁴, NR⁴R⁴, CF₃,CH₂OH, CH₂F, CH₂Cl, CH₂CF₃, C(Y³)₃, C(Y³)₂C(Y³)₃, C(═O)OH, C(═O)OR⁴,C(═O)-alkyl, C(═O)-aryl, C(═O)-alkoxyalkyl, C(═O)NH₂, C(═O)NHR⁴,C(═O)N(R⁴)₂, where each Y³ is independently H or halo; and each R⁴independently is H, acyl including lower acyl, alkyl including loweralkyl such as but not limited to methyl, ethyl, propyl and cyclopropyl,alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxyalkyl, hydroxyalkyl, oraryl.

In a subembodiment, R₆ is ethyl, CH₂CH₂OH, or CH₂-phenyl.

In another embodiment, the triciribine compounds provided herein havethe following structure:

wherein R₇ is H, halo, alkyl (including lower alkyl), alkenyl, alkynyl,alkoxy, alkoxyalkyl, hydroxyalkyl, cycloalkyl, nitro, cyano, OH, OR⁴,NH₂, NHR⁴, NR⁴R⁴, SH, SR⁴, CF₃, CH₂OH, CH₂F, CH₂Cl, CH₂CF₃, C(Y³)₃,C(Y³)₂C(Y³)₃, C(═O)OH, C(═O)OR⁴, C(═O)-alkyl, C(═O)-aryl,C(═O)-alkoxyalkyl, C(═O)NH₂, C(═O)NHR⁴, C(═O)N(R⁴)₂, or N₃, where eachY³ is independently H or halo; and each R⁴ independently is H, acylincluding lower acyl, alkyl including lower alkyl such as but notlimited to methyl, ethyl, propyl and cyclopropyl, alkenyl, alkynyl,cycloalkyl, alkoxy, alkoxyalkyl, hydroxyalkyl.

In a subembodiment, R₇ is methyl, ethyl, phenyl, chloro or NH₂.

In another embodiment, the triciribine compounds provided herein havethe following structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

As used herein and unless otherwise indicated, the term “taxanes” refersto a compound of formula II:

wherein

R¹⁰ and R¹¹ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, aryl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In one embodiment, the taxane compound has the following structure:

wherein R¹¹ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, aryl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In another embodiment, the taxane compound has the following structure:

wherein R¹¹ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, aryl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In another embodiment, the taxane compound has the following structure:

In another embodiment, the taxane compound has the following structure:

It is to be understood that the compounds disclosed herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. It is understood that the disclosure of acompound herein encompasses any racemic, optically active, polymorphic,or steroisomeric form, or mixtures thereof, which preferably possessesthe useful properties described herein, it being well known in the arthow to prepare optically active forms and how to determine activityusing the standard tests described herein, or using other similar testswhich are will known in the art.

Examples of methods that can be used to obtain optical isomers of thecompounds include the following:

(i) physical separation of crystals—a technique whereby macroscopiccrystals of the individual enantiomers are manually separated. Thistechnique can be used if crystals of the separate enantiomers exist,i.e., the material is a conglomerate, and the crystals are visuallydistinct;

(ii) simultaneous crystallization—a technique whereby the individualenantiomers are separately crystallized from a solution of the racemate,possible only if the latter is a conglomerate in the solid state;

(iii) enzymatic resolutions—a technique whereby partial or completeseparation of a racemate by virtue of differing rates of reaction forthe enantiomers with an enzyme

(iv) enzymatic asymmetric synthesis—a synthetic technique whereby atleast one step of the synthesis uses an enzymatic reaction to obtain anenantiomerically pure or enriched synthetic precursor of the desiredenantiomer;

(v) chemical asymmetric synthesis—a synthetic technique whereby thedesired enantiomer is synthesized from an achiral precursor underconditions that produce assymetry (i.e., chirality) in the product,which may be achieved using chiral catalysts or chiral auxiliaries;

(vi) diastereomer separations—a technique whereby a racemic compound isreacted with an enantiomerically pure reagent (the chiral auxiliary)that converts the individual enantiomers to diastereomers. The resultingdiastereomers are then separated by chromatography or crystallization byvirtue of their now more distinct structural differences and the chiralauxiliary later removed to obtain the desired enantiomer;

(vii) first- and second-order asymmetric transformations—a techniquewhereby diastereomers from the racemate equilibrate to yield apreponderance in solution of the diastereomer from the desiredenantiomer or where preferential crystallization of the diastereomerfrom the desired enantiomer perturbs the equilibrium such thateventually in principle all the material is converted to the crystallinediastereomer from the desired enantiomer. The desired enantiomer is thenreleased from the diastereomer;

(viii) kinetic resolutions—this technique refers to the achievement ofpartial or complete resolution of a racemate (or of a further resolutionof a partially resolved compound) by virtue of unequal reaction rates ofthe enantiomers with a chiral, non-racemic reagent or catalyst underkinetic conditions;

(ix) enantiospecific synthesis from non-racemic precursors—a synthetictechnique whereby the desired enantiomer is obtained from non-chiralstarting materials and where the stereochemical integrity is not or isonly minimally compromised over the course of the synthesis;

(x) chiral liquid chromatography—a technique whereby the enantiomers ofa racemate are separated in a liquid mobile phase by virtue of theirdiffering interactions with a stationary phase. The stationary phase canbe made of chiral material or the mobile phase can contain an additionalchiral material to provoke the differing interactions;

(xi) chiral gas chromatography—a technique whereby the racemate isvolatilized and enantiomers are separated by virtue of their differinginteractions in the gaseous mobile phase with a column containing afixed non-racemic chiral adsorbent phase;

(xii) extraction with chiral solvents—a technique whereby theenantiomers are separated by virtue of preferential dissolution of oneenantiomer into a particular chiral solvent;

(xiii) transport across chiral membranes—a technique whereby a racemateis placed in contact with a thin membrane barrier. The barrier typicallyseparates two miscible fluids, one containing the racemate, and adriving force such as concentration or pressure differential causespreferential transport across the membrane barrier. Separation occurs asa result of the non-racemic chiral nature of the membrane which allowsonly one enantiomer of the racemate to pass through.

In some embodiments, triciribine, triciribine phosphate (TCN-P),triciribine 5′-phosphate (TCN-P), or the DMF adduct of triciribine(TCN-DMF) are provided. TCN can be synthesized by any technique known toone skilled in the art, for example, as described in TetrahedronLetters, vol. 49, pp. 4757-4760 (1971). TCN-P can be prepared by anytechnique known to one skilled in the art, for example, as described inU.S. Pat. No. 4,123,524. The synthesis of TCN-DMF is described, forexample, in INSERM, vol. 81, pp. 37-82 (1978). Other compounds relatedto TCN as described herein can be synthesized, for example, according tothe methods disclosed in Gudmundsson, K. S., et al., “Synthesis ofcarbocyclic analogs of 2′,3′-dideoxysangivamycin,2′,3′-dideoxytoyocamycin, and 2′,3′-dideoxytriciribine,” NucleosidesNucleotides Nucleic Acids, 20(10-11): 1823-1830 (October-November 2001);Porcari, A. R., et al., “6-N-Acyltriciribine analogues:structure-activity relationship between acyl carbon chain length andactivity against HIV-1,” J. Med. Chem., 43(12):2457-2463 (Jun. 15,2000); Porcari, A. R., et al., “Acyclic sugar analogs of triciribine:lack of antiviral and antiproliferative activity correlate with lowintracellular phosphorylation,” Nucleosides Nucleotides,18(11-12):2475-2497 (November-December 1999), Porcari, A. R., et al.,“Deoxy sugar analogues of triciribine: correlation of antiviral andantiproliferative activity with intracellular phosphorylation,” J. Med.Chem., 43(12):2438-2448 (Jun. 15, 2000), Porcari, A. R., et al.,“Synthesis and antiviral activity of 2-substituted analogs oftriciribine,” Nucleosides Nucleotides Nucleic Acids, 22(12):2171-2193(December 2003), Porcari, A. R., et al., “An improved total synthesis oftriciribine: a tricyclic nucleoside with antineoplastic and antiviralproperties,” Nucleosides Nucleotides Nucleic Acids, 23(1-2):31-39(2004), Schweinsberg, P. D., et al. “Identification of the metabolitesof an antitumor tricyclic nucleoside (NSC-154020),” Biochem. Pharmacol.,30(18):2521-2526 (Sep. 15, 1981), Smith, K. L., et al., “Synthesis ofnew 2′-beta-C-methyl related triciribine analogues as anti-HCV agents,”Bioorg. Med. Chem. Lett., 14(13):3517-3520 (Jul. 5, 2004), Townsend, L.B., et al., “The synthesis and biological activity of certainpentaazaacenaphthylenes, hexaazaacenaphthylenes and their correspondingnucleosides,” Nucleic Acids Symp. Ser., 1986(17):41-44 (1986), and/orWotring, L. L., et al., “Mechanism of activation of triciribinephosphate (TCN-P) as a prodrug form of TCN,” Cancer Treat Rep.,70(4):491-7 (April 1986).

5.3. Pharmaceutically Acceptable Salts and Prodrugs

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compound as apharmaceutically acceptable salt may be appropriate. Pharmaceuticallyacceptable salts include those derived from pharmaceutically acceptableinorganic or organic bases and acids. Suitable salts include thosederived from alkali metals such as potassium and sodium, alkaline earthmetals such as calcium and magnesium, among numerous other acids wellknown in the pharmaceutical art. In particular, examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids, which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including, sulfate,nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

Any of the nucleotides described herein can be administered as anucleotide prodrug to increase the activity, bioavailability, stabilityor otherwise alter the properties of the nucleoside. A number ofnucleotide prodrug ligands are known. In general, alkylation, acylationor other lipophilic modification of the mono, di or triphosphate of thenucleoside will increase the stability of the nucleotide. Examples ofsubstituent groups that can replace one or more hydrogens on thephosphate moiety are alkyl, aryl, steroids, carbohydrates, includingsugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jonesand N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of thesecan be used in combination with the disclosed nucleosides to achieve adesired effect.

In one embodiment, the triciribine or a related compound is provided as5′-hydroxyl lipophilic prodrug. Nonlimiting examples of U.S. patentsthat disclose suitable lipophilic substituents that can be covalentlyincorporated into the nucleoside, preferably at the 5′-OH position ofthe nucleoside or lipophilic preparations, include U.S. Pat. No.5,149,794 (Sep. 22, 1992, Yatvin, et al.); U.S. Pat. No. 5,194,654 (mar.16, 1993, Hostetler, et al.); U.S. Pat. No. 5,223,263 (Jun. 29, 1993,Hostetler, et al.); U.S. Pat. No. 5,256,641 (Oct. 26, 1993, Yatvin, etal.); U.S. Pat. No. 5,411,947 (May 2, 1995, Hostetler, et al.); U.S.Pat. No. 5,463,092 (Oct. 31, 1995, Hostetler, et al.); U.S. Pat. No.5,543,389 (Aug. 6, 1996, Yatvin, et al.); U.S. Pat. No. 5,543,390 (Aug.6, 1996, Yatvin, et al.); U.S. Pat. No. 5,543,391 (Aug. 6, 1996, Yatvin,et al.); and U.S. Pat. No. 5,554,728 (Sep. 10, 1996, Basava, et al.),all of which are incorporated herein by reference.

Foreign patent applications that disclose lipophilic substituents thatcan be attached to the triciribine or a related compound s of thepresent invention, or lipophilic preparations, include WO 89/02733, WO90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO/15132,EP 0 350 287, EP 93917054.4, and WO 91/19721.

Additional nonlimiting examples of derivatives of triciribine or arelated compounds are those that contain substituents as described inthe following publications. These derivatized triciribine or a relatedcompound s can be used for the indications described in the text orotherwise as antiviral agents, including as anti-HIV or anti-HBV agents.Ho, D. H. W. (1973) Distribution of Kinase and deaminase of1β-D-arabinofuranosylcytosine in tissues of man and mouse. Cancer Res.33, 2816-2820; Holy, A. (1993) Isopolar phosphorous-modified nucleotideanalogues. In: De Clercq (ed.), Advances in Antiviral Drug Design, Vol.I, JAI Press, pp. 179-231; Hong, C. I., Nechaev, A., and West, C. R.(1979a) Synthesis and antitumor activity of1β-3-arabinofuranosylcytosine conjugates of cortisol and cortisone.Biochem. Biophys. Rs. Commun. 88, 1223-1229; Hong, C. I., Nechaev, A.,Kirisits, A. J. Buchheit, D. J. and West, C. R. (1980) Nucleosideconjugates as potential antitumor agents. 3. Synthesis and antitumoractivity of 1-(β-D-arabinofuranosyl)cytosine conjugates ofcorticosteriods and selected lipophilic alcohols. J. Med. Chem. 28,171-177; Hostetler, K. Y., Stuhmiller, L. M., Lenting, H. B. M. van denBosch, H. and Richman, D. D. (1990) Synthesis and antiretroviralactivity of phospholipid analogs of azidothymidine and other antiviralnucleosides. J Biol. Chem. 266, 11714-11717; Hostetler, K. Y., Korba, B.Sridhar, C., Gardener, M. (1994a) Antiviral activity ofphosphatidyl-dideoxycytidine in hepatitis B-infected cells and enhancedhepatic uptake in mice. Antiviral Res. 24, 59-67; Hostetler, K. Y.,Richman, D. D., Sridhar, C. N. Felgner, P. L., Felgner, J., Ricci, J.,Gerdener, M. F. Selleseth, D. W. and Ellis, M. N. (1994b)Phosphatidylazidothymidine and phosphatidyl-ddC: Assessment of uptake inmouse lymphoid tissues and antiviral activities in humanimmunodeficiency virus-infected cells and in rauscher leukemiavirus-infected mice. Antimicrobial Agents Chemother. 38, 2792-2797;Hunston, R. N., Jones, A. A. McGuigan, C., Walker, R. T., Balzarini, J.,and De Clercq, E. (1984) Synthesis and biological properties of somecyclic phosphotriesters derived from 2′-deoxy-5-fluorouridine. J. Med.Chem. 27, 440-444; Ji, Y. H., Moog, C., Schmitt, G., Bischoff, P. andLuu, B. (1990); Monophosphoric acid diesters of 73-hydroxycholesteroland of pyrimidine nucleosides as potential antitumor agents; synthesisand preliminary evaluation of antitumor activity. J. Med. Chem. 33,2264-2270; Jones, A. S., McGuigan, C., Walter, R. T., Balzarini, J. andDeClercq, E. (1984) Synthesis, properties, and biological activity ofsome nucleoside cyclic phosphoramidates. J. Chem. Soc. Perkin Trans. I,1471-1474; Juodka, B. A. and Smart, J. (1974) Synthesis ofditribonucleoside a (P N) amino acid derivatives. Coll. Czech. Chem.Comm. 39, 363-968; Kataoka, S., Imai, J., Yamaji, N., Kato, M., Saito,M., Kawada, T. and Imai, S. 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(1969). Toxicological assessment and health standardratings for ethylene sulfide in the industrial atmosphere. Gig. Trf.Prof. Zabol. 13, 47-48 (Chem. Abstr. 72, 212); Robins, R. K. (1984) Thepotential of nucleotide analogs as inhibitors of retroviruses andtumors. Pharm. Res. 11-18; Rosowsky, A., Kim, S. H., Ross and J. Wick,M. M. (1982) Lipophilic 5′-(alkylphosphate) esters of1-β-D-arabinofuranosylcytosine and its N⁴-acyl and 2.2′-anhydro-3′0-acylderivatives as potential prodrugs. J. Med. Chem. 25, 171-178; Ross, W.(1961) Increased sensitivity of the walker turnout towards aromaticnitrogen mustards carrying basic side chains following glucosepretreatment. Biochem. Pharm. 8, 235-240; Ryu, E. K., Ross, R. J.,Matsushita, T., MacCoss, M., Hong, C. I. and West, C. R. (1982).Phospholipid-nucleoside conjugates 3. Synthesis and preliminarybiological evaluation of 1-β-D-arabinofuranosylcytosine5′diphosphate[-], 2-diacylglycerols. J. Med. Chem. 25, 1322-1329;Saffhill, R. and Hume, W. J. (1986) The degradation of5-iododeoxyurindine and 5-bromoeoxyuridine by serum from differentsources and its consequences for the use of these compounds forincorporation into DNA. Chem. Biol. Interact. 57, 347-355; Saneyoshi,M., Morozumi, M., Kodama, K., Machida, J., Kuninaka, A. and Yoshino, H.(1980) Synthetic nucleosides and nucleotides XVI. Synthesis andbiological evaluations of a series of 1-β-D-arabinofuranosylcytosine5′-alkyl or arylphosphates. Chem. Pharm. Bull. 28, 2915-2923; Sastry, J.K., Nehete, P. N., Khan, S., Nowak, B. J., Plunkett, W., Arlinghaus, R.B. and Farquhar, D. (1992) Membrane-permeable dideoxyuridine5′-monophosphate analogue inhibits human immunodeficiency virusinfection. Mol. Pharmacol. 41, 441-445; Shaw, J. P., Jones, R. J.Arimilli, M. N., Louie, M. S., Lee, W. A. and Cundy, K. C. (1994) Oralbioavailability of PMEA from PMEA prodrugs in male Sprague-Dawley rats.9th Annual AAPS Meeting. San Diego, Calif. (Abstract). Shuto, S., Ueda,S., Imamura, S., Fukukuawa, K. Matsuda, A. and Ueda, T. (1987) A facileone-step synthesis of 5′-phosphatidylnucleosides by an enzymatictwo-phase reaction. Tetrahedron Lett. 28, 199-202; Shuto, S., Itoh, H.,Ueda, S., Imamura, S., Kukukawa, K., Tsujino, M. Matsuda, A. and Ueda,T. (1988) A facile enzymatic synthesis of5′-(3-sn-phosphatidyl)nucleosides and their antileukemic activities.Chem. Pharm. Bull. 36, 209-217. One preferred phosphate prodrug group isthe S-acyl-2-thioethyl group, also referred to as “SATE.”

Additional examples of prodrugs that can be used are those described inthe following patents and patent applications: U.S. Pat. Nos. 5,614,548,5,512,671, 5,770,584, 5,962,437, 5,223,263, 5,817,638, 6,252,060,6,448,392, 5,411,947, 5,744,592, 5,484,809, 5,827,831, 5,696,277,6,022,029, 5,780,617, 5,194,654, 5,463,092, 5,744,461, 4,444,766,4,562,179, 4,599,205, 4,493,832, 4,221,732, 5,116,992, 6,429,227,5,149,794, 5,703,063, 5,888,990, 4,810,697, 5,512,671, 6,030,960,2004/0259845, 6,670,341, 2004/0161398, 2002/082242, 5,512,671,2002/0082242, and or PCT Publication Nos WO 90/11079, WO 96/39197,and/or WO 93/08807.

5.4. In Vivo Efficacy/Dosing Regimens

In another aspect of the present invention, dosing regimens are providedthat limit the toxic side effects of TCN and related compounds. In oneembodiment, such dosing regimens minimize the following toxic sideeffects, including, but not limited to, hepatoxicity, thrombocytopenia,hyperglycemia, vomiting, hypocalcemia, anemia, hypoalbunemia,myelosuppression, hypertriglyceridemia, hyperamylasemia, diarrhea,stomachitis and/or fever.

In another embodiment, the administration of TCN, TCN-P or relatedcompounds and one or more taxanes provides at least a partial orcomplete response in vivo in at least 15-20% of the subjects. Inparticular embodiments, a partial response can be at least 15, 20, 25,30, 35, 40, 50, 55, 60, 65, 70, 75, 80 or 85% regression of the tumor.In other embodiments, this response can be evident in at least 15, 15,20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85 or 90% of thesubjects treated with the therapy. In further embodiments, such responserates can be obtained by any therapeutic regimen disclosed herein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with cancer by administering to the subject an effectiveamount of TCN, TCN-P or a related compound and one or more taxanesaccording to a dosing schedule that includes administering thetriciribine compound and/or the taxane one time per week for three weeksfollowed by a one week period wherein the drug is not administered(i.e., via a 28 day cycle). In other embodiments, such 28 day cycles canbe repeated at least 2, 3, 4, or 5 times or until regression of thetumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the triciribine compoundand/or the taxane is not administered. In other embodiments, such 42 daycycles can be repeated at least 2, 3, 4, or 5 times or until regressionof the tumor is evident. In a particular embodiment, less than 12, lessthan 11 or less than 10 mg/m² of TCN, TCN-P or a related compound can beadministered according to a 42 day cycle. In other particularembodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mg/m² of TCN, TCN-P or arelated compound can be administered according to a 42 day cycle. Inanother particular embodiment, about 1 to about 50 mg of one or moretaxanes is administered. In a particular embodiment, 1, 5, 10, 15, 20,25, 30, 35, or 40 mg of one or more taxanes can be administeredaccording to a 42 day cycle.

In another embodiment, methods are provided to treat cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P or a related compound and less than about 30 mg of one ormore taxanes one time per week. In particular embodiments, 0.5, 1, 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10mg/m² of TCN, TCN-P or a related compound as disclosed herein can beadministered one time per week In another particular embodiment, 1, 5,10, 15, 20, 25, 30, 35, or 40 mg of one or more taxanes can beadministered one time per week.

In embodiments of the present invention, the compounds disclosed hereincan be administered simultaneously as a single bolus dose over a shortperiod of time, for example, about 5, 10, 15, 20, 30 or 60 minutes. Infurther embodiments, dosing schedules are provided in which thecompounds are administered simultaneously via continuous infusion for atleast 24, 48, 72, 96, or 120 hours. In certain embodiments, theadministration of the triciribine compound and/or the taxane viacontinuous or bolus injections can be repeated at a certain frequency atleast: once a week, once every two weeks, once every three weeks, once amonth, once every five weeks, once every six weeks, once every eightweeks, once every ten weeks and/or once every twelve weeks. The type andfrequency of administrations can be combined in any manner disclosedherein to create a dosing cycle. The triciribine compound and/or thetaxane can be repeatedly administered via a certain dosing cycles, forexample as a bolus injection once every two weeks for three months. Thedosing cycles can be administered for at least: one, two three, fourfive, six, seven, eight, nine, ten, eleven, twelve, eighteen or twentyfour months. Alternatively, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,15 or 20 dosing cycles can be administered to a patient. The triciribinecompound and/or the taxane can be administered according to anycombination disclosed herein, for example, the triciribine compoundand/or the taxane can be administered once a week every three weeks for3 cycles.

In further embodiments, the compounds can be administered separately atleast once a day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which thetriciribine compound and/or the taxane are not administered.

The TCN, TCN-P and related compounds and one or more taxanes asdisclosed herein can be administered to patients in an amount that iseffective in causing tumor regression. The administration of TCN, TCN-Por related compounds and one or more taxanes can provide at least apartial, such as at least 15, 20 or 30%, or complete response in vivo inat least 15-20% of the subjects. In certain embodiments, at least 2, 5,10, 15, 20, 30 or 50 mg/m² of a triciribine compound disclosed hereincan be administered to a subject. In certain embodiments, at least about0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 165, 175, 200, 250, 300, or 350 mg/m² of TCN,TCN-P or a related compound disclosed herein can be administered to asubject. In certain embodiments, 1, 5, 10, 15, 20, 25, 30, 35, or 40 mgof one or more taxanes can be administered to a subject.

The administration of the compound can be conducted according to any oftherapeutic regimens disclosed herein. In particular embodiments, thedosing regimen includes administering less than about 20 mg/m² of TCNand related compounds and less than about 30 mg of one or more taxaneseither concurrently, sequentially, or conducted over a period of time.In one embodiment, less than 20 mg/m² of TCN or related compounds can beadministered once a week concurrently with less than about 30 mg of oneor more taxanes. In another embodiment, less than 20 mg/m² of TCN orrelated compounds can be administered once a week and less than about 30mg of one or more taxanes can be administered the following week.

In further embodiments, 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m² ofTCN or a related compound and less than about 30, 25, 20, 15, or 10 mgof one or more taxanes can be administered to a subject. In anotherembodiment, less than 10 mg/m² of a triciribine compound and less thanabout 30 mg of one or more taxanes can be administered to a subject viacontinuous infusion for at least five days. The present inventionprovides for any combination of dosing type, frequency, number of cyclesand dosage amount disclosed herein.

5.5. Screening of Patient Populations

In another aspect of the present invention, methods are provided toidentify cancers or tumors that are susceptible to the toxic effects oftriciribine (TCN) and related compounds. In one embodiment, methods areprovided to treat a cancer or tumor in a mammal by (i) obtaining abiological sample from the tumor; (ii) determining whether the cancer ortumor overexpresses Akt kinase or hyperactivated and phosphorylated Aktkinase, and (iii) treating the cancer or tumor with triciribine or arelated compound as described herein. In one embodiment, the biologicalsample can be a biopsy. In other embodiments, the biological sample canbe fluid, cells and/or aspirates obtained from the tumor or cancer.

The biological sample can be obtained according to any technique knownto one skilled in the art. In one embodiment, a biopsy can be conductedto obtain the biological sample. A biopsy is a procedure performed toremove tissue or cells from the body for examination. Some biopsies canbe performed in a physician's office, while others need to be done in ahospital setting. In addition, some biopsies require use of ananesthetic to numb the area, while others do not require any sedation.In certain embodiments, an endoscopic biopsy can be performed. This typeof biopsy is performed through a fiberoptic endoscope (a long, thin tubethat has a close-focusing telescope on the end for viewing) through anatural body orifice (i.e., rectum) or a small incision (i.e.,arthroscopy). The endoscope is used to view the organ in question forabnormal or suspicious areas, in order to obtain a small amount oftissue for study. Endoscopic procedures are named for the organ or bodyarea to be visualized and/or treated. The physician can insert theendoscope into the gastrointestinal tract (alimentary tract endoscopy),bladder (cystoscopy), abdominal cavity (laparoscopy), joint cavity(arthroscopy), mid-portion of the chest (mediastinoscopy), or tracheaand bronchial system (laryngoscopy and bronchoscopy).

In another embodiment, a bone marrow biopsy can be performed. This typeof biopsy can be performed either from the sternum (breastbone) or theiliac crest hipbone (the bone area on either side of the pelvis on thelower back area). The skin is cleansed and a local anesthetic is givento numb the area. A long, rigid needle is inserted into the marrow, andcells are aspirated for study; this step is occasionally uncomfortable.A core biopsy (removing a small bone ‘chip’ from the marrow) may followthe aspiration.

In a further embodiment, an excisional or incisional biopsy can beperformed on the mammal. This type of biopsy is often used when a wideror deeper portion of the skin is needed. Using a scalpel (surgicalknife), a full thickness of skin is removed for further examination, andthe wound is sutured (sewed shut with surgical thread). When the entiretumor is removed, it is referred to as an excisional biopsy technique.If only a portion of the tumor is removed, it is referred to as anincisional biopsy technique. Excisional biopsy is often the methodusually preferred, for example, when melanoma (a type of skin cancer) issuspected.

In still further embodiments, a fine needle aspiration (FNA) biopsy canbe used. This type of biopsy involves using a thin needle to remove verysmall pieces from a tumor. Local anesthetic is sometimes used to numbthe area, but the test rarely causes much discomfort and leaves no scar.FNA is not, for example, used for diagnosis of a suspicious mole, butmay be used, for example, to biopsy large lymph nodes near a melanoma tosee if the melanoma has metastasized (spread). A computed tomographyscan (CT or CAT scan) can be used to guide a needle into a tumor in aninternal organ such as the lung or liver.

In other embodiments, punch shave and/or skin biopsies can be conducted.Punch biopsies involve taking a deeper sample of skin with a biopsyinstrument that removes a short cylinder, or “apple core,” of tissue.After a local anesthetic is administered, the instrument is rotated onthe surface of the skin until it cuts through all the layers, includingthe dermis, epidermis, and the most superficial parts of the subcutis(fat). A shave biopsy involves removing the top layers of skin byshaving it off. Shave biopsies are also performed with a localanesthetic. Skin biopsies involve removing a sample of skin forexamination under the microscope to determine if, for example, melanomais present. The biopsy is performed under local anesthesia.

In particular embodiment, methods are provided to determine whether thetumor overexpresses an Akt kinase. Akt kinase overexpression can referto the phosphorylation state of the kinase. Hyperphosphorylation of Aktcan be detected according to the methods described herein. In oneembodiment, a tumor biopsy can be compared to a control tissue. Thecontrol tissue can be a normal tissue from the mammal in which thebiopsy was obtained or a normal tissue from a healthy mammal. Akt kinaseoverexpression or hyperphosphorylation can be determined if the tumorbiopsy contains greater amounts of Akt kinase and/or Akt kinasephosphorylation than the control tissue, such as, for example, at leastapproximately 1.5, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5,4.75, 5, 5.5, 6, 7, 8, 9, or 10-fold greater amounts of Akt kinase thancontained in the control tissue.

In one embodiment, the present invention provides a method to detectaberrant Akt kinase expression in a subject or in a biological samplefrom the subject by contacting cells, cell extracts, serum or othersample from the subjects or said biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formation, wherein an elevated presence ofthe complex relative to a normal cell is indicative of an aberrant cellthat expresses or overexpresses Akt. In one example, cells or cellextracts can be screened immunologically for the presence of elevatedlevels of Akt kinase.

In an alternative embodiment, the aberrant expression of Akt in a cellis detected at the genetic level by screening for the level ofexpression of a gene encoding an Akt kinase wherein an elevated level ofa transcriptional expression product (i.e., mRNA) compared to a normalcell is indicative of an aberrant cell. In certain embodiments,real-time PCR as well as other PCR procedures can be used to determinetranscriptional activity. In one embodiment, mRNA can be obtained fromcells of a subject or from a biological sample from a subject and cDNAoptionally generated. The mRNA or cDNA can then be contacted with agenetic probe capable of hybridizing to and/or amplifying all or part ofa nucleotide sequence encoding Akt kinase or its complementarynucleotide sequence and then the level of the mRNA or cDNA can bedetected wherein the presence of elevated levels of the mRNA or cDNAcompared to normal controls can be assessed.

Yet another embodiment of the present invention contemplates the use ofan antibody, monoclonal or polyclonal, to Akt kinase in a quantitativeor semi-quantitative diagnostic kit to determine relative levels of Aktkinase in suspected cancer cells from a patient, which can include allthe reagents necessary to perform the assay. In one embodiment, a kitutilizing reagents and materials necessary to perform an ELISA assay isprovided. Reagents can include, for example, washing buffer, antibodydilution buffer, blocking buffer, cell staining solution, developingsolution, stop solution, anti-phospho-protein specific antibodies,anti-Pan protein specific antibodies, secondary antibodies, anddistilled water. The kit can also include instructions for use and canoptionally be automated or semi-automated or in a form which iscompatible with automated machine or software. In one embodiment, aphosphor-ser-473 Akt antibody that detects the activated form of AKT(Akt phosphorylated at serine 474) can be utilized as the antibody in adiagnostic kit. See, for example, Yuan et al. (2000) “FrequentActivation of AKT2 and induction of apoptosis by inhibition ofphosphinositide-3-OH kinase/Akt pathway in human ovarian cancer,”Oncogene 19:2324-2330.

5.6. Akt Kinases

Akt, also named PKB³, represents a subfamily of the serine/threoninekinase. Three members, AKT1, AKT2, and AKT3, have been identified inthis subfamily. Akt is activated by extracellular stimuli in aPI3K-dependent manner (Datta, S. R., et al. Genes Dev. 13: 2905-2927,1999). Full activation of Akt requires phosphorylation of Thr³⁰⁸ in theactivation loop and Ser⁴⁷³ in the C-terminal activation domain. Akt isnegatively regulated by PTEN tumor suppressor. Mutations in PTEN havebeen identified in various tumors, which lead to activation of Aktpathway (Datta, S. R., et al. Genes Dev. 13: 2905-2927, 1999). Inaddition, amplification, overexpression and/or activation of Akt havebeen detected in a number of human malignancies (Datta, S. R., et al.Genes Dev. 13: 2905-2927, 1999, Cheng, J. Q., and Nicosia, S. V. AKTsignal transduction pathway in oncogenesis. In Schwab D, editor.Encyclopedic Reference of Cancer. Berlin Heidelberg and New York:Springer; 2001. pp 35-7). Ectopic expression of Akt, especiallyconstitutively active Akt, induces cell survival and malignanttransformation whereas inhibition of Akt activity stimulates apoptosisin a range of mammalian cells (Datta, S. R., et al. Genes Dev. 13:2905-2927, 1999, Cheng, J. Q., and Nicosia, S. V. AKT signaltransduction pathway in oncogenesis. In Schwab D, editor. EncyclopedicReference of Cancer. Berlin Heidelberg and New York: Springer; 2001. pp35-7, Sun, M., et al. Am. J. Path., 159: 431-437, 2001, Cheng, J. Q., etal. Oncogene, 14: 2793-2801, 1997). Further, activation of Akt has beenshown to associate with tumor invasiveness and chemoresistance (West, K.A., et al. Drug Resist. Updat., 5: 234-248, 2002).

Activation of the Akt pathway plays a pivotal role in malignanttransformation and chemoresistance by inducing cell survival, growth,migration, and angiogenesis. The present invention provides methods todetermine levels of Akt kinase overexpression and/or hyperactivated andphosphorylated Akt kinase.

The Akt kinase can be any known Akt family kinase, or kinase relatedthereto, including, but not limited to Akt 1, Akt 2, Akt 3. The mRNA andamino acid sequences of human Akt1, Akt2, and Akt 3 are illustrated inFIGS. 6 a-c, 7a-d, and 8a-c, respectively.

In another embodiment, the compositions of the invention including TCN,TCN-P or related compounds and one or more taxanes kills cancer or tumorcells which express Akt.

5.7. Diagnostic Assays

Immunological Assays

In one embodiment, a method is provided for detecting the aberrantexpression of an Akt kinase in a cell in a mammal or in a biologicalsample from the mammal, by contacting cells, cell extracts or serum orother sample from the mammal or biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formations and determining whether anelevated presence of the complex relative to a normal cell is present.

The immunointeractive molecule can be a molecule having specificity andbinding affinity for an Akt kinase or its antigenic parts or itshomologs or derivatives thereof. In one embodiment, theimmunointeractive molecule can be an immunoglobulin molecule. In otherembodiments, the immunointeractive molecules can be an antibodyfragments, single chain antibodies, and/or deimmunized moleculesincluding humanized antibodies and T-cell associated antigen-bindingmolecules (TABMs). In one particular embodiment, the antibody can be amonoclonal antibody. In another particular embodiment, the antibody canbe a polyclonal antibody. The immunointeractive molecule can exhibitspecificity for an Akt kinase or more particularly an antigenicdeterminant or epitope on an Akt kinase. An antigenic determinant orepitope on an Akt kinase includes that part of the molecule to which animmune response is directed. The antigenic determinant or epitope can bea B-cell epitope or where appropriate a T-cell epitope. In oneembodiment, the antibody is a phosphor-ser 473 Akt antibody.

One embodiment of the present invention provides a method for diagnosingthe presence of cancer or cancer-like growth in a mammal, in whichaberrant Akt activity is present, by contacting cells or cell extractsfrom the mammal or a biological sample from the subject with an Aktkinase-binding effective amount of an antibody having specificity forthe Akt kinase or an antigenic determinant or epitope thereon and thenquantitatively or qualitatively determining the level of an Aktkinase-antibody complex wherein the presence of elevated levels of saidcomplex compared to a normal cell is determined.

Antibodies can be prepared by any of a number of means known to oneskilled in the art. For example, for the detection of human Akt kinase,antibodies can be generally but not necessarily derived from non-humananimals such as primates, livestock animals (e.g. sheep, cows, pigs,goats, horses), laboratory test animals (e.g. mice, rats, guinea pigs,rabbits) and/or companion animals (e.g. dogs, cats). Antibodies may alsobe recombinantly produced in prokaryotic or eukaryotic host cells.Generally, antibody based assays can be conducted in vitro on cell ortissue biopsies. However, if an antibody is suitably deimmunized or, inthe case of human use, humanized, then the antibody can be labeled with,for example, a nuclear tag, administered to a patient and the site ofnuclear label accumulation determined by radiological techniques. TheAkt kinase antibody can be a cancer targeting agent. Accordingly,another embodiment of the present invention provides deimmunized formsof the antibodies for use in cancer imaging in human and non-humanpatients.

In general, for the generation of antibodies to an Akt kinase, theenzyme is required to be extracted from a biological sample whether thisbe from animal including human tissue or from cell culture if producedby recombinant means. The Akt kinase can be separated from thebiological sample by any suitable means. For example, the separation maytake advantage of any one or more of the Akt kinase's surface chargeproperties, size, density, biological activity and its affinity foranother entity (e.g. another protein or chemical compound to which itbinds or otherwise associates). Thus, for example, separation of the Aktkinase from the biological fluid can be achieved by any one or more ofultra-centrifugation, ion-exchange chromatography (e.g. anion exchangechromatography, cation exchange chromatography), electrophoresis (e.g.polyacrylamide gel electrophoresis, isoelectric focussing), sizeseparation (e.g., gel filtration, ultra-filtration) andaffinity-mediated separation (e.g. immunoaffinity separation including,but not limited to, magnetic bead separation such as Dynabead(trademark) separation, immunochromatography, immuno-precipitation). Theseparation of Akt kinase from the biological fluid can preserveconformational epitopes present on the kinase and, thus, suitably avoidstechniques that cause denaturation of the enzyme. In a furtherembodiment, the kinase can be separated from the biological fluid usingany one or more of affinity separation, gel filtration and/orultra-filtration.

Immunization and subsequent production of monoclonal antibodies can becarried out using standard protocols known in the art, such as, forexample, described by Kohler and Milstein (Kohler and Milstein, Nature256: 495-499, 1975; Kohler and Milstein, Eur. J. Immunol. 6(7): 511-519,1976), Coligan et al. (“Current Protocols in Immunology, John Wiley &Sons, Inc., 1991-1997) or Toyama et al. (Monoclonal Antibody, ExperimentManual”, published by Kodansha Scientific, 1987). Essentially, an animalis immunized with an Akt kinase-containing biological fluid or fractionthereof or a recombinant form of Akt kinase by standard methods toproduce antibody-producing cells, particularly antibody-producingsomatic cells (e.g. B lymphocytes). These cells can then be removed fromthe immunized animal for immortalization. In certain embodiment, afragment of an Akt kinase can be used to the generate antibodies. Thefragment can be associated with a carrier. The carrier can be anysubstance of typically high molecular weight to which a non- or poorlyimmunogenic substance (e.g. a hapten) is naturally or artificiallylinked to enhance its immunogenicity.

Immortalization of antibody-producing cells can be carried out usingmethods which are well-known in the art. For example, theimmortalization may be achieved by the transformation method usingEpstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology 121: 140,1986). In another embodiment, antibody-producing cells are immortalizedusing the cell fusion method (described in Coligan et al., 1991-1997,supra), which is widely employed for the production of monoclonalantibodies. In this method, somatic antibody-producing cells with thepotential to produce antibodies, particularly B cells, are fused with amyeloma cell line. These somatic cells may be derived from the lymphnodes, spleens and peripheral blood of primed animals, preferably rodentanimals such as mice and rats. In a particular embodiment, mice spleencells can be used. In other embodiments, rat, rabbit, sheep or goatcells can also be used. Specialized myeloma cell lines have beendeveloped from lymphocytic tumours for use in hybridoma-producing fusionprocedures (Kohler and Milstein, 1976, supra; Shulman et al., Nature276: 269-270, 1978; Volk et al., J. Virol. 42(1): 220-227, 1982). Manymyeloma cell lines can also be used for the production of fused cellhybrids, including, e.g. P3.times.63-Ag8, P3.times.63-AG8.653,P3/NS1-Ag4-1 (NS-1), Sp2/0-Ag14 and S194/5.XXO.Bu.1. The P3.times.63-Ag8and NS-1 cell lines have been described by Kohler and Milstein (1976,supra). Shulman et al. (1978, supra) developed the Sp2/0-Ag14 myelomaline. The S194/5.XXO.Bu.1 line was reported by Trowbridge (J. Exp. Med.148(1): 313-323, 1978). Methods for generating hybrids ofantibody-producing spleen or lymph node cells and myeloma cells usuallyinvolve mixing somatic cells with myeloma cells in a 10:1 proportion(although the proportion may vary from about 20:1 to about 1:1),respectively, in the presence of an agent or agents (chemical, viral orelectrical) that promotes the fusion of cell membranes. Fusion methodshave been described (Kohler and Milstein, 1975, supra; Kohler andMilstein, 1976, supra; Gefter et al., Somatic Cell Genet. 3: 231-236,1977; Volk et al., 1982, supra). The fusion-promoting agents used bythose investigators were Sendai virus and polyethylene glycol (PEG). Incertain embodiments, means to select the fused cell hybrids from theremaining unfused cells, particularly the unfused myeloma cells, areprovided. Generally, the selection of fused cell hybrids can beaccomplished by culturing the cells in media that support the growth ofhybridomas but prevent the growth of the unfused myeloma cells, whichnormally would go on dividing indefinitely. The somatic cells used inthe fusion do not maintain long-term viability in in vitro culture andhence do not pose a problem. Several weeks are required to selectivelyculture the fused cell hybrids. Early in this time period, it isnecessary to identify those hybrids which produce the desired antibody,so that they may subsequently be cloned and propagated. Generally,around 10% of the hybrids obtained produce the desired antibody,although a range of from about 1 to about 30% is not uncommon. Thedetection of antibody-producing hybrids can be achieved by any one ofseveral standard assay methods, including enzyme-linked immunoassay andradioimmunoassay techniques as, for example, described in Kennet et al.(Monoclonal Antibodies and Hybridomas: A New Dimension in BiologicalAnalyses, pp 376-384, Plenum Press, New York, 1980) and by FACS analysis(O'Reilly et al., Biotechniques 25: 824-830, 1998).

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line may bepropagated in either of two standard ways. A suspension of the hybridomacells can be injected into a histocompatible animal. The injected animalwill then develop tumours that secrete the specific monoclonal antibodyproduced by the fused cell hybrid. The body fluids of the animal, suchas serum or ascites fluid, can be tapped to provide monoclonalantibodies in high concentration. Alternatively, the individual celllines may be propagated in vitro in laboratory culture vessels. Theculture medium containing high concentrations of a single specificmonoclonal antibody can be harvested by decantation, filtration orcentrifugation, and subsequently purified.

The cell lines can then be tested for their specificity to detect theAkt kinase of interest by any suitable immunodetection means. Forexample, cell lines can be aliquoted into a number of wells andincubated and the supernatant from each well is analyzed byenzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibodytechnique, or the like. The cell line(s) producing a monoclonal antibodycapable of recognizing the target LIM kinase but which does notrecognize non-target epitopes are identified and then directly culturedin vitro or injected into a histocompatible animal to form tumours andto produce, collect and purify the required antibodies.

The present invention provides, therefore, a method of detecting in asample an Akt kinase or fragment, variant or derivative thereofincluding contacting the sample with an antibody or fragment orderivative thereof and detecting the level of a complex containing theantibody and Akt kinase or fragment, variant or derivative thereofcompared to normal controls wherein elevated levels of Akt kinase isdetermined. Any suitable technique for determining formation of thecomplex may be used. For example, an antibody according to theinvention, having a reporter molecule associated therewith, may beutilized in immunoassays. Such immunoassays include but are not limitedto radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs)immunochromatographic techniques (ICTs), and Western blotting which arewell known to those of skill in the art. Immunoassays can also includecompetitive assays. The present invention encompasses qualitative andquantitative immunoassays.

Suitable immunoassay techniques are described, for example, in U.S. Pat.Nos. 4,016,043; 4,424,279; and 4,018,653. These include both single-siteand two-site assays of the non-competitive types, as well as thetraditional competitive binding assays. These assays also include directbinding of a labeled antigen-binding molecule to a target antigen.

The invention further provides methods for quantifying Akt proteinexpression and activation levels in cells or tissue samples obtainedfrom an animal, such as a human cancer patient or an individualsuspected of having cancer. In one embodiment, the invention providesmethods for quantifying Akt protein expression or activation levelsusing an imaging system quantitatively. The imaging system can be usedto receive, enhance, and process images of cells or tissue samples, thathave been stained with AKT protein-specific stains, in order todetermine the amount or activation level of AKT protein expressed in thecells or tissue samples from such an animal. In embodiments of themethods of the invention, a calibration curve of AKT1 and AKT2 proteinexpression can be generated for at least two cell lines expressingdiffering amounts of AKT protein. The calibration curve can then used toquantitatively determine the amount of AKT protein that is expressed ina cell or tissue sample. Analogous calibration curves can be made foractivated AKT proteins using reagents specific for the activationfeatures. It can also be used to determine changes in amounts andactivation state of AKT before and after clinical cancer treatment.

In one particular embodiment of the methods of the invention, AKTprotein expression in a cell or tissue sample can be quantified using anenzyme-linked immunoabsorbent assay (ELISA) to determine the amount ofAKT protein in a sample. Such methods are described, for example, inU.S. Patent Publication No. 2002/0015974.

In other embodiments enzyme immunoassays can be used to detect the Aktkinase. In such assays, an enzyme is conjugated to the second antibody,generally by means of glutaraldehyde or periodate. The substrates to beused with the specific enzymes are generally chosen for the productionof, upon hydrolysis by the corresponding enzyme, a detectable colourchange. It is also possible to employ fluorogenic substrates, whichyield a fluorescent product rather than the chromogenic substrates. Theenzyme-labeled antibody can be added to the first antibody-antigencomplex, allowed to bind, and then the excess reagent washed away. Asolution containing the appropriate substrate can then be added to thecomplex of antibody-antigen-antibody. The substrate can react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of antigenwhich was present in the sample. Alternately, fluorescent compounds,such as fluorescein, rhodamine and the lanthanide, europium (EU), can bechemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic colour visually detectable with a lightmicroscope. The fluorescent-labeled antibody is allowed to bind to thefirst antibody-antigen complex. After washing off the unbound reagent,the remaining tertiary complex is then exposed to light of anappropriate wavelength. The fluorescence observed indicates the presenceof the antigen of interest. Immunofluorometric assays (IFMA) are wellestablished in the art and are particularly useful for the presentmethod. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules can also be employed.

In a particular embodiment, antibodies to Akt kinase can also be used inELISA-mediated detection of Akt kinase especially in serum or othercirculatory fluid. This can be accomplished by immobilizing anti-Aktkinase antibodies to a solid support and contacting these with abiological extract such as serum, blood, lymph or other bodily fluid,cell extract or cell biopsy. Labeled anti-Akt kinase antibodies can thenbe used to detect immobilized Akt kinase. This assay can be varied inany number of ways and all variations are encompassed by the presentinvention and known to one skilled in the art. This approach can enablerapid detection and quantitation of Akt kinase levels using, forexample, a serum-based assay.

In one embodiment, an Akt Elisa assay kit may be used in the presentinvention. For example, a Cellular Activation of Signaling ELISA kit forAkt S473 from SuperArray Bioscience can be utilized in the presentinvention. In one embodiment, the antibody can be an anti-pan antibodythat recognizes Akt S473. Elisa assay kit containing an anti-Aktantibody and additional reagents, including, but not limited to, washingbuffer, antibody dilution buffer, blocking buffer, cell stainingsolution, developing solution, stop solution, secondary antibodies, anddistilled water.

Nucleotide Detection

In another embodiment, a method to detect Akt kinases is provided bydetecting the level of expression in a cell of a polynucleotide encodingan Akt kinase. Expression of the polynucleotide can be determined usingany suitable technique known to one skilled in the art. In oneembodiment, a labeled polynucleotide encoding an Akt kinase can beutilized as a probe in a Northern blot of an RNA extract obtained fromthe cell. In other embodiments, a nucleic acid extract from an animalcan be utilized in concert with oligonucleotide primers corresponding tosense and antisense sequences of a polynucleotide encoding the kinase,or flanking sequences thereof, in a nucleic acid amplification reactionsuch as RT PCR. A variety of automated solid-phase detection techniquesare also available to one skilled in the art, for example, as describedby Fodor et al. (Science 251: 767-777, 1991) and Kazal et al. (NatureMedicine 2: 753-759, 1996).

In other embodiments, methods are provided to detect akt kinase encodingRNA transcripts. The RNA can be isolated from a cellular samplesuspected of containing Akt kinase RNA, e.g. total RNA isolated fromhuman cancer tissue. RNA can be isolated by methods known in the art,e.g. using TRIZOL reagent (GIBCO-BRL/Life Technologies, Gaithersburg,Md.). Oligo-dT, or random-sequence oligonucleotides, as well assequence-specific oligonucleotides can be employed as a primer in areverse transcriptase reaction to prepare first-strand cDNAs from theisolated RNA. Resultant first-strand cDNAs can then amplified withsequence-specific oligonucleotides in PCR reactions to yield anamplified product.

Polymerase chain reaction or “PCR” refers to a procedure or technique inwhich amounts of a preselected fragment of nucleic acid, RNA and/or DNA,are amplified as described, for example, in U.S. Pat. No. 4,683,195.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers. These primerswill be identical or similar in sequence to opposite strands of thetemplate to be amplified. PCR can be used to amplify specific RNAsequences and cDNA transcribed from total cellular RNA. See generallyMullis et al. (Quant. Biol. 51: 263, 1987; Erlich, eds., PCR Technology,Stockton Press, NY, 1989). Thus, amplification of specific nucleic acidsequences by PCR relies upon oligonucleotides or “primers” havingconserved nucleotide sequences wherein the conserved sequences arededuced from alignments of related gene or protein sequences, e.g. asequence comparison of mammalian Akt kinase genes. For example, oneprimer is prepared which is predicted to anneal to the antisense strandand another primer prepared which is predicted to anneal to the sensestrand of a cDNA molecule which encodes a Akt kinase. To detect theamplified product, the reaction mixture is typically subjected toagarose gel electrophoresis or other convenient separation technique andthe relative presence of the Akt kinase specific amplified DNA detected.For example, Akt kinase amplified DNA may be detected using Southernhybridization with a specific oligonucleotide probe or comparing itselectrophoretic mobility with DNA standards of known molecular weight.Isolation, purification and characterization of the amplified Akt kinaseDNA can be accomplished by excising or eluting the fragment from the gel(for example, see references Lawn et al., Nucleic Acids Res. 2: 6103,1981; Goeddel et al., Nucleic cids Res. 8: 4057-1980), cloning theamplified product into a cloning site of a suitable vector, such as thepCRII vector (Invitrogen), sequencing the cloned insert and comparingthe DNA sequence to the known sequence of LIM kinase. The relativeamounts of LIM kinase mRNA and cDNA can then be determined.

In one embodiment, real-time PCR can be used to determinetranscriptional levels of Akt nucleotides. Determination oftranscriptional activity also includes a measure of potentialtranslational activity based on available mRNA transcripts. Real-timePCR as well as other PCR procedures use a number of chemistries fordetection of PCR product including the binding of DNA bindingfluorophores, the 5′ endonuclease, adjacent liner and hairpinoligoprobes and the self-fluorescing amplicons. These chemistries andreal-time PCR in general are discussed, for example, in Mackay et al.,Nucleic Acids Res 30(6): 1292-1305, 2002; Walker, J. Biochem. Mol.Toxicology 15(3): 121-127, 2001; Lewis et al., J. Pathol. 195: 66-71,2001.

In an alternate embodiment, the aberrant expression of Akt can beidentified by contacting a nucleotide sequences isolated from abiological sample with an oligonucleotide probe having a sequencecomplementary to an Akt sequences selected from the nucleotide sequencesof FIGS. 6 a-c, 7 a-d, or 8 a-c, or fragment thereof, and then detectingthe sequence by hybridizing the probe to the sequence, and comparing theresults to a normal sample. The hybridization of the probe to thebiological sample can be detected by labeling the probe using anydetectable agent. The probe can be labeled for example, with aradioisotope, or with biotin, fluorescent dye, electron-dense reagent,enzyme, hapten or protein for which antibodies are available. Thedetectable label can be assayed by any desired means, includingspectroscopic, photochemical, biochemical, immunochemical,radioisotopic, or chemical means. The probe can also be detected usingtechniques such as an oligomer restriction technique, a dot blot assay,a reverse dot blot assay, a line probe assay, and a 5′ nuclease assay.Alternatively, the probe can be detected using any of the generallyapplicable DNA array technologies, including macroarray, microarray andDNA microchip technologies. The oligonucleotide probe typically includesapproximately at least 14, 15, 16, 18, 20, 25 or 28 nucleotides thathybridize to the nucleotides selected from FIGS. 6 a-c, 7 a-d, and 8a-c, or a fragment thereof. It is generally not preferred to use a probethat is greater than approximately 25 or 28 nucleotides in length. Theoligonucleotide probe is designed to identify an Akt nucleotidesequence.

Kinase Assays

The activity of the Akt kinases can be measured using any suitablekinase assay known in the art. For example, and not by way oflimitation, the methods described in Hogg et al (Oncogene 1994 9:98-96),Mills et al (J. Biol. Chem. 1992 267:16000-006) and Tomizawa et al 2001(FEBS Lett. 2001 492: 221-7), Schmandt et al, (J. Immunol. 1994,152:96-105) can be used. Further serine, threonine and tyrosine kinaseassays are described in Ausubel et al. (Short Protocols in MolecularBiology, 1999, unit 17.6).

Akt kinase assays can generally use an Akt polypeptide, a labeled donorsubstrate, and a receptor substrate that is either specific ornon-specific for Akt. In such assays Akt transfers a labeled moiety fromthe donor substrate to the receptor substrate, and kinase activity ismeasured by the amount of labeled moiety transferred from the donorsubstrate to the receptor substrate. Akt polypeptide can be producedusing various expression systems, can be purified from cells, can be inthe form of a cleaved or uncleaved recombinant fusion protein and/or canhave non-Akt polypeptide sequences, for example a His tag or.beta.-galactosidase at its N- or C-terminus. Akt activity can beassayed in cancerous cells lines if the cancerous cell lines are used asa source of the Akt to be assayed. Suitable donor substrates for Aktassays include any molecule that is susceptible to dephosphorylation byAkt., such as, for example include .gamma.-labeled ATP and ATP analogs,wherein the label is ³³P, ³²P, ³⁵S or any other radioactive isotope or asuitable fluorescent marker. Suitable recipient substrates for Aktassays include any polypeptide or other molecule that is susceptible tophosphorylation by Akt. Recipient substrates can be derived fromfragments of in vivo targets of Akt. Recipient substrates fragments canbe 8 to 50 amino acids in length, usually 10 to 30 amino acids andparticularly of about 10, 12, 15, 18, 20 and 25 amino acids in length.Further recipient substrates can be determined empirically using a setof different polypeptides or other molecules. Targets of Recipientsubstrates for TTK can be capable of being purified from othercomponents of the reaction once the reaction has been performed. Thispurification is usually done through a molecular interaction, where therecipient substrates is biotinylated and purified through itsinteraction with streptavidin, or a specific antibody is available thatcan specifically recognize the recipient substrates. The reaction can beperformed in a variety of conditions, such as on a solid support, in agel, in solution or in living cells. The choice of detection methodsdepends on type of label used for the donor molecule and may include,for example, measurement of incorporated radiation or fluorescence byautoradiography, scintillation, scanning or fluorography.

6. METHODS OF TREATMENT

The compounds and pharmaceutical compositions provided herein can beused in the treatment of a condition including tumors, cancer, and otherdisorders associated with abnormal cell proliferation. In oneembodiment, the compounds of the present invention can be used to treata carcinoma, sarcoma, lymphoma, leukemia, and/or myeloma. In otherembodiments of the present invention, the compounds disclosed herein canbe used to treat solid tumors.

The compounds of the present invention can be used for the treatment ofcancer, such as, but not limited to cancer of the following organs ortissues: breast, prostate, lung, bronchus, colon, urinary, bladder,non-Hodgkin lymphoma, melanoma, kidney, renal, pancreas, pharnx,thyroid, stomach, brain, multiple myeloma, esophagus, liver,intrahepatic bile duct, cervix, larynx, acute myeloid leukemia, chroniclymphatic leukemia, soft tissue, such as heart, Hodgkin lymphoma,testis, small intestine, chronic myeloid leukemia, acute lymphaticleukemia, anus, anal canal, anorectal, thyroid, vulva, gallbladder,pleura, eye, nose nasal cavity, middle ear, nasopharnx, ureter,peritoneum, omentum, mesentery, and gastrointestineal, high gradeglioma, glioblastoma, colon, rectal, pancreatic, gastric cancers,hepatocellular carcinoma; head and neck cancers, carcinomas; renal cellcarcinoma; adenocarcinoma; sarcomas; hemangioendothelioma; lymphomas;leukemias, mycosis fungoides. In additional embodiments, the compoundsof the present invention can be used to treat skin diseases including,but not limited to, the malignant diseases angiosarcoma,hemangioendothelioma, basal cell carcinoma, squamous cell carcinoma,malignant melanoma and Kaposi's sarcoma, and the non-malignant diseasesor conditions such as psoriasis, lymphangiogenesis, hemangioma ofchildhood, Sturge-Weber syndrome, verruca vulgaris, neurofibromatosis,tuberous sclerosis, pyogenic granulomas, recessive dystrophicepidermolysis bullosa, venous ulcers, acne, rosacea, eczema, molluscumcontagious, seborrheic keratosis, and actinic keratosis.

Compositions including the compounds of the invention can be used totreat these cancers and other cancers at any stage from the discovery ofthe cancer to advanced stages. In addition, compositions includingcompounds of the invention can be used in the treatment of the primarycancer and metastases thereof.

In other embodiments of the invention, the compounds described hereincan be used for the treatment of cancer, including, but not limited to,the cancers listed in Table 1 below.

TABLE 1 Types of Cancer Acute Lymphoblastic Leukemia, Hairy CellLeukemia Adult Head and Neck Cancer Acute Lymphoblastic Leukemia,Hepatocellular (Liver) Cancer, Childhood Adult (Primary) Acute MyeloidLeukemia, Adult Hepatocellular (Liver) Cancer, Acute Myeloid Leukemia,Childhood (Primary) Childhood Hodgkin's Lymphoma, Adult AdrenocorticalCarcinoma Hodgkin's Lymphoma, Childhood Adrenocortical Carcinoma,Hodgkin's Lymphoma During Childhood Pregnancy AIDS-Related CancersHypopharyngeal Cancer AIDS-Related Lymphoma Hypothalamic and VisualPathway Anal Cancer Glioma, Childhood Astrocytoma, Childhood CerebellarIntraocular Melanoma Astrocytoma, Childhood Cerebral Islet CellCarcinoma (Endocrine Basal Cell Carcinoma Pancreas) Bile Duct Cancer,Extrahepatic Kaposi's Sarcoma Bladder Cancer Kidney (Renal Cell) CancerBladder Cancer, Childhood Kidney Cancer, Childhood Bone Cancer,Laryngeal Cancer Osteosarcoma/Malignant Fibrous Laryngeal Cancer,Childhood Histiocytoma Leukemia, Acute Lymphoblastic, Brain Stem Glioma,Childhood Adult Brain Tumor, Adult Leukemia, Acute Lymphoblastic, BrainTumor, Brain Stem Glioma, Childhood Childhood Leukemia, Acute Myeloid,Adult Brain Tumor, Cerebellar Leukemia, Acute Myeloid, Astrocytoma,Childhood Childhood Brain Tumor, Cerebral Leukemia, Chronic LymphocyticAstrocytoma/Malignant Glioma, Leukemia, Chronic Myelogenous ChildhoodLeukemia, B Cell Brain Tumor, Ependymoma, Lip and Oral Cavity CancerChildhood Liver Cancer, Adult (Primary) Brain Tumor, Medulloblastoma,Liver Cancer, Childhood (Primary) Childhood Lung Cancer, Non-Small CellBrain Tumor, Supratentorial Lung Cancer, Small Cell PrimitiveNeuroectodermal Lymphoma, AIDS-Related Tumors, Childhood Lymphoma,Burkitt's Brain Tumor, Visual Pathway and Lymphoma, Cutaneous T-Cell,see Hypothalamic Glioma, Childhood Mycosis Fungoides and Sézary BrainTumor, Childhood Syndrome Breast Cancer Lymphoma, Hodgkin's, AdultBreast Cancer, Childhood Lymphoma, Hodgkin's, Childhood Breast Cancer,Male Lymphoma, Hodgkin's During Bronchial Adenomas/Carcinoids, PregnancyChildhood Lymphoma, Non-Hodgkin's, Adult Burkitt's Lymphoma Lymphoma,Non-Hodgkin's, Carcinoid Tumor, Childhood Childhood Carcinoid Tumor,Gastrointestinal Lymphoma, Non-Hodgkin's During Carcinoma of UnknownPrimary Pregnancy Central Nervous System Lymphoma, Primary CentralLymphoma, Primary Nervous System Cerebellar Astrocytoma, ChildhoodMacroglobulinemia, Waldenström's Cerebral Astrocytoma/MalignantMalignant Fibrous Histiocytoma of Glioma, Childhood Bone/OsteosarcomaCervical Cancer Medulloblastoma, Childhood Childhood Cancers MelanomaChronic Lymphocytic Leukemia Melanoma, Intraocular (Eye) ChronicMyelogenous Leukemia Merkel Cell Carcinoma Chronic MyeloproliferativeMesothelioma, Adult Malignant Disorders Mesothelioma, Childhood ColonCancer Metastatic Squamous Neck Cancer Colorectal Cancer, Childhood withOccult Primary Cutaneous T-Cell Lymphoma, see Multiple EndocrineNeoplasia Mycosis Fungoides and Sézary Syndrome, Childhood SyndromeMultiple Myeloma/Plasma Cell Endometrial Cancer Neoplasm Ependymoma,Childhood Mycosis Fungoides Esophageal Cancer Myelodysplastic SyndromesEsophageal Cancer, Childhood Myelodysplastic/Myeloproliferative Ewing'sFamily of Tumors Diseases Extracranial Germ Cell Tumor, MyelogenousLeukemia, Chronic Childhood Myeloid Leukemia, Adult Acute ExtragonadalGerm Cell Tumor Myeloid Leukemia, Childhood Extrahepatic Bile DuctCancer Acute Eye Cancer, Intraocular Melanoma Myeloma, Multiple EyeCancer, Retinoblastoma Myeloproliferative Disorders, Gallbladder CancerChronic Gastric (Stomach) Cancer Nasal Cavity and Paranasal SinusGastric (Stomach) Cancer, Cancer Childhood Nasopharyngeal CancerGastrointestinal Carcinoid Tumor Nasopharyngeal Cancer, Childhood GermCell Tumor, Extracranial, Neuroblastoma Childhood Non-Hodgkin'sLymphoma, Adult Germ Cell Tumor, Extragonadal Non-Hodgkin's Lymphoma,Germ Cell Tumor, Ovarian Childhood Gestational Trophoblastic TumorNon-Hodgkin's Lymphoma Glioma, Adult During Pregnancy Glioma, ChildhoodBrain Stem Non-Small Cell Lung Cancer Glioma, Childhood Cerebral OralCancer, Childhood Astrocytoma Oral Cavity Cancer, Lip and Glioma,Childhood Visual Pathway Oropharyngeal Cancer and HypothalamicOsteosarcoma/Malignant Fibrous Skin Cancer (Melanoma) Histiocytoma ofBone Skin Carcinoma, Merkel Cell Ovarian Cancer, Childhood Small CellLung Cancer Ovarian Epithelial Cancer Small Intestine Cancer OvarianGerm Cell Tumor Soft Tissue Sarcoma, Adult Ovarian Low MalignantPotential Soft Tissue Sarcoma, Childhood Tumor Squamous Cell Carcinoma,see Pancreatic Cancer Skin Cancer (non-Melanoma) Pancreatic Cancer,Childhood Squamous Neck Cancer with Pancreatic Cancer, Islet Cell OccultPrimary, Metastatic Paranasal Sinus and Nasal Cavity Stomach (Gastric)Cancer Cancer Stomach (Gastric) Cancer, Parathyroid Cancer ChildhoodPenile Cancer Supratentorial Primitive Pheochromocytoma NeuroectodermalTumors, Pineoblastoma and Supratentorial Childhood PrimitiveNeuroectodermal T-Cell Lymphoma, Cutaneous, see Tumors, ChildhoodMycosis Fungoides and Sézary Pituitary Tumor Syndrome Plasma CellNeoplasm/Multiple Testicular Cancer Myeloma Thymoma, ChildhoodPleuropulmonary Blastoma Thymoma and Thymic Carcinoma Pregnancy andBreast Cancer Thyroid Cancer Pregnancy and Hodgkin's Thyroid Cancer,Childhood Lymphoma Transitional Cell Cancer of the Pregnancy andNon-Hodgkin's Renal Pelvis and Ureter Lymphoma Trophoblastic Tumor,Gestational Primary Central Nervous System Unknown Primary Site,Carcinoma Lymphoma of, Adult Prostate Cancer Unknown Primary Site,Cancer of, Rectal Cancer Childhood Renal Cell (Kidney) Cancer UnusualCancers of Childhood Renal Cell (Kidney) Cancer, Ureter and RenalPelvis, Childhood Transitional Cell Cancer Renal Pelvis and Ureter,Urethral Cancer Transitional Cell Cancer Uterine Cancer, EndometrialRetinoblastoma Uterine Sarcoma Rhabdomyosarcoma, Childhood VaginalCancer Salivary Gland Cancer Visual Pathway and Hypothalamic SalivaryGland Cancer, Childhood Glioma, Childhood Sarcoma, Ewing's Family ofVulvar Cancer Tumors Waldenström's Macroglobulinemia Sarcoma, Kaposi'sWilms' Tumor Sarcoma, Soft Tissue, Adult Sarcoma, Soft Tissue, ChildhoodSarcoma, Uterine Sezary Syndrome Skin Cancer (non-Melanoma) Skin Cancer,Childhood

In further embodiments of the present invention, the compounds disclosedherein can be used in the treatment of angiogenesis-related diseases.

Antiangiogenic small molecules include thalidomide, which acts in partby inhibiting NFkB, 2-methoxyestradiol, which influences microtubuleactivation and hypoxia inducing factor (HIF1a) activation,cyclo-oxygenase 2 (COX2) inhibitors, and low doses of conventionalchemotherapeutic agents, including cyclophosphamide, taxanes, and vincaalkaloids (vincristine, vinblastine) (D'Amato, R. J. et al. (1994) Proc.Natl. Acad. Sci. U.S.A 91, 3964-3968, D'Amato, R. J. et al. (1994) Proc.Natl. Acad. Sci. U.S.A 91, 4082-4085). In addition, certain tyrosinekinase inhibitors indirectly decrease angiogenesis by decreasingproduction of VEGF and other proangiogenic factors by tumor and stromalcells. These drugs include Herceptin, imatinib (Glivec), and Iressa(Bergers, G. et al. (2003) Journal of Clinical Investigation 111,1287-1295, Ciardiello, F. et al. (2001) Clinical Cancer Research 7,1459-1465, Plum, S. M. et al. (2003) Clinical Cancer Research 9,4619-4626).

Recently, angiogenesis inhibitors have moved from animal models to humanpatients. Angiogenesis inhibitors represent a promising treatment for avariety of cancers. Recently, Avastin a high affinity antibody againstvascular endothelial growth factor (VEGF), has been shown to prolonglife as a single agent in advanced renal cell carcinoma and prolong lifein combination with chemotherapy in advanced colon cancer (Yang, J. C.et al. (2003) New England Journal of Medicine 349, 427-434, Kabbinavar,F. et al. (2003) Journal of Clinical Oncology 21, 60-65).

Angiogenesis-related diseases include, but are not limited to,inflammatory, autoimmune, and infectious diseases;angiogenesis-dependent cancer, including, for example, solid tumors,blood born tumors such as leukemias, and tumor metastases; benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis;eczema; ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rubeosis;Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. In addition, compositions of this invention canbe used to treat diseases such as, but not limited to, intestinaladhesions, atherosclerosis, scleroderma, warts, and hypertrophic scars(i.e., keloids). Compositions of this invention can also be used in thetreatment of diseases that have angiogenesis as a pathologic consequencesuch as cat scratch disease (Rochele minalia quintosa), ulcers(Helobacter pylori), tuberculosis, and leprosy.

6.1. Treatment of Drug Resistant Tumors or Cancers

The invention provides compounds that can be used to treat drugresistant cancer, including the embodiments of cancers and thetriciribine compound and/or the taxane disclosed herein.

Multidrug resistance (MDR) occurs in human cancers and can be asignificant obstacle to the success of chemotherapy. Multidrugresistance is a phenomenon whereby tumor cells in vitro that have beenexposed to one cytotoxic agent develop cross-resistance to a range ofstructurally and functionally unrelated compounds. In addition, MDR canoccur intrinsically in some cancers without previous exposure tochemotherapy agents. Thus, in one embodiment, the present inventionprovides methods for the treatment of a patient with a drug resistantcancer, for example, multidrug resistant cancer, by administration ofTCN, TCN-P or a related compound and one or more taxanes as disclosedherein. In certain embodiments, TCN, TCN-P and related compounds and oneor more taxanes can be used to treat cancers that are resistant to taxolalone, rapamycin, tamoxifen, cisplatin, and/or gefitinib (iressa).

In one embodiment, TCN, TCN-P or a related compound and one or moretaxanes as disclosed herein can be used for the treatment of drugresistant cancers of the colon, bone, kidney, adrenal, pancreas, liverand/or any other cancer known in the art or described herein.

6.2. Combination Therapy

In one embodiment, the triciribine compounds and taxanes of theinvention can be administered together with other cytotoxic agents. Inanother embodiment, the triciribine compounds and one or more taxanesand compositions thereof, when used in the treatment of solid tumors,can be administered in conjunction with the use of radiation.

In another embodiment of the present invention, the triciribinecompounds and taxanes and compositions disclosed herein can be combinedwith at least one additional chemotherapeutic agent. The additionalagents can be administered in combination or alternation with thecompounds disclosed herein. The drugs can form part of the samecomposition, or be provided as a separate composition for administrationat the same time or a different time.

In one embodiment, the triciribine compounds and taxanes disclosedherein can be combined with antiangiogenic agents to enhance theireffectiveness, or combined with other antiangiogenic agents andadministered together with other cytotoxic agents. In anotherembodiment, the triciribine compounds and taxanes and compositions, whenused in the treatment of solid tumors, can be administered with theagents selected from, but not limited to IL-12, retinoids, interferons,angiostatin, endostatin, thalidomide, thrombospondin-1,thrombospondin-2, captopryl, antineoplastic agents such as alphainterferon, COMP (cyclophosphamide, vincristine, methotrexate andprednisone), etoposide, mBACOD (methortrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP(prednisone, methotrexate (w/leucovin rescue), doxorubicin,cyclophosphamide, etoposide/mechlorethamine, vincristine, prednisone andprocarbazine), vincristine, vinblastine, angioinhibins, TNP-470,pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101,CM-101, Techgalan, thalidomide, SP-PG and radiation. In furtherembodiments, the compounds and compositions disclosed herein can beadministered in combination or alternation with, for example, drugs withantimitotic effects, such as those which target cytoskeletal elements,including podophylotoxins or vinca alkaloids (vincristine, vinblastine);antimetabolite drugs (such as 5-fluorouracil, cytarabine, gemcitabine,purine analogues such as pentostatin, methotrexate); alkylating agentsor nitrogen mustards (such as nitrosoureas, cyclophosphamide orifosphamide); drugs which target DNA such as the antracycline drugsadriamycin, doxorubicin, pharmorubicin or epirubicin; drugs which targettopoisomerases such as etoposide; hormones and hormone agonists orantagonists such as estrogens, antiestrogens (tamoxifen and relatedcompounds) and androgens, flutamide, leuprorelin, goserelin, cyprotroneor octreotide; drugs which target signal transduction in tumour cellsincluding antibody derivatives such as herceptin; alkylating drugs suchas platinum drugs (cisplatin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed below in Table 2.

TABLE 2 Chemotherapeutic Agents 13-cis-Retinoic Acid Neosar 2-Amino-6-Neulasta Mercaptopurine Neumega 2-CdA Neupogen 2-ChlorodeoxyadenosineNilandron 5-fluorouracil Nilutamide 5-FU Nitrogen Mustard 6-TG Novaldex6-Thioguanine Novantrone 6-Mercaptopurine Octreotide 6-MP Octreotideacetate Accutane Oncospar Actinomycin-D Oncovin Adriamycin Ontak AdrucilOnxal Agrylin Oprevelkin Ala-Cort Orapred Aldesleukin OrasoneAlemtuzumab Oxaliplatin Alitretinoin Paclitaxel Alkaban-AQ PamidronateAlkeran Panretin All-transretinoic acid Paraplatin Alpha interferonPediapred Altretamine PEG Interferon Amethopterin PegaspargaseAmifostine Pegfilgrastim Aminoglutethimide PEG-INTRON AnagrelidePEG-L-asparaginase Anandron Phenylalanine Mustard Anastrozole PlatinolArabinosylcytosine Platinol-AQ Ara-C Prednisolone Aranesp Prednis oneAredia Prelone Arimidex Procarbazine Aromasin PROCRIT Arsenic trioxideProleukin Asparaginase Prolifeprospan 20 with Carmustine implant ATRAPurinethol Avastin Raloxifene BCG Rheumatrex BCNU Rituxan BevacizumabRituximab Bexarotene Roveron-A (interferon alfa-2a) Bicalutamide RubexBiCNU Rubidomycin hydrochloride Blenoxane Sandostatin BleomycinSandostatin LAR Bortezomib Sargramostim Busulfan Solu-Cortef BusulfexSolu-Medrol C225 STI-571 Calcium Leucovorin Streptozocin CampathTamoxifen Camptosar Targretin Camptothecin-11 Taxol CapecitabineTaxotere Carac Temodar Carboplatin Temozolomide Carmustine TeniposideCarmustine wafer TESPA Casodex Thalidomide CCNU Thalomid CDDP TheraCysCeeNU Thioguanine Cerubidine Thioguanine Tabloid cetuximabThiophosphoamide Chlorambucil Thioplex Cisplatin Thiotepa CitrovorumFactor TICE Cladribine Toposar Cortisone Topotecan Cosmegen ToremifeneCPT-11 Trastuzumab Cyclophosphamide Tretinoin Cytadren TrexallCytarabine Trisenox Cytarabine liposomal TSPA Cytosar-U VCR CytoxanVelban Dacarbazine Velcade Dactinomycin VePesid Darbepoetin alfaVesanoid Daunomycin Viadur Daunorubicin Vinblastine DaunorubicinVinblastine Sulfate hydrochloride Vincasar Pfs Daunorubicin liposomalVincristine DaunoXome Vinorelbine Decadron Vinorelbine tartrateDelta-Cortef VLB Deltasone VP-16 Denileukin diftitox Vumon DepoCytXeloda Dexamethasone Zanosar Dexamethasone acetate Zevalin dexamethasonesodium Zinecard phosphate Zoladex Dexasone Zoledronic acid DexrazoxaneZometa DHAD Gliadel wafer DIC Glivec Diodex GM-CSF Docetaxel GoserelinDoxil granulocyte-colony stimulating factor Doxorubicin Granulocytemacrophage colony stimulating Doxorubicin liposomal factor DroxiaHalotestin DTIC Herceptin DTIC-Dome Hexadrol Duralone Hexalen EfudexHexamethylmelamine Eligard HMM Ellence Hycamtin Eloxatin Hydrea ElsparHydrocort Acetate Emcyt Hydrocortisone Epirubicin Hydrocortisone sodiumphosphate Epoetin alfa Hydrocortisone sodium succinate ErbituxHydrocortone phosphate Erwinia L-asparaginase Hydroxyurea EstramustineIbritumomab Ethyol Ibritumomab Tiuxetan Etopophos Idamycin EtoposideIdarubicin Etoposide phosphate Ifex Eulexin IFN-alpha Evista IfosfamideExemestane IL-2 Fareston IL-11 Faslodex Imatinib mesylate FemaraImidazole Carboxamide Filgrastim Interferon alfa Floxuridine InterferonAlfa-2b (PEG conjugate) Fludara Interleukin-2 Fludarabine Interleukin-11Fluoroplex Intron A (interferon alfa-2b) Fluorouracil LeucovorinFluorouracil (cream) Leukeran Fluoxymesterone Leukine FlutamideLeuprolide Folinic Acid Leurocristine FUDR Leustatin FulvestrantLiposomal Ara-C G-CSF Liquid Pred Gefitinib Lomustine Gemcitabine L-PAMGemtuzumab ozogamicin L-Sarcolysin Gemzar Meticorten Gleevec MitomycinLupron Mitomycin-C Lupron Depot Mitoxantrone Matulane M-PrednisolMaxidex MTC Mechlorethamine MTX Mechlorethamine Mustargen HydrochlorineMustine Medralone Mutamycin Medrol Myleran Megace Iressa MegestrolIrinotecan Megestrol Acetate Isotretinoin Melphalan KidrolaseMercaptopurine Lanacort Mesna L-asparaginase Mesnex LCR MethotrexateMethotrexate Sodium Methylprednisolone Mylocel Letrozole

In certain embodiments, interferons (IFNs) can be used in combinationswith the compounds of the present invention. Suitable intereferonsinclude: interferon alpha-2a, interferon alpha-2b, pegylated interferonalpha, including interferon alpha-2a and interferon alpha 2b, interferonbeta, interferon gamma, interferon tau, interferon omega, INFERGEN(interferon alphacon-1) by InterMune, OMNIFERON (natural interferon) byViragen, ALBUFERON by Human Genome Sciences, REBIF (interferon beta-1a)by Ares-Serono, Omega Interferon by BioMedicine, Oral Interferon Alphaby Amarillo Biosciences, and interferon gamma, interferon tau, and/orinterferon gamma-1b by InterMune.

In one embodiment TCN, TCN-P or a related compound and taxanes asdisclosed herein can be used in combination or alternation withadditional chemotherapeutic agents, such as those described herein or inTable 3, for the treatment of drug resistant cancer, for examplemultiple drug resistant cancer. Drug resistant cancers can includecancers of the colon, bone, kidney, adrenal, pancreas, liver and/or anyother cancer known in the art or described herein. In one embodiment,the additional chemotherapeutic agent can be a P-glycoprotein inhibitor.In certain non-limiting embodiments, the P-glycoprotein inhibitor can beselected from the following drugs: verapamil, cyclosporin (such ascyclosporin A), tamoxifen, calmodulin antagonists, dexverapamil,dexniguldipine, valspodar (PSC 833), biricodar (VX-710), tariquidar(XR9576), zosuquidar (LY335979), laniquidar (R101933), and/or ONT-093.

7. PHARMACEUTICAL COMPOSITIONS

The compositions including triciribine compounds and one or more taxanescan optionally be administered with a pharmaceutical carrier orexcipient. Pharmaceutical carriers suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. The triciribine compounds and in combination with one ormore taxanes may be formulated as the sole pharmaceutically activeingredient in the composition or may be combined with one or moretaxanes.

Compositions including the triciribine compounds and one or more taxanesmay be suitable for oral, rectal, nasal, topical (including buccal andsublingual), vaginal, or parenteral (including subcutaneous,intramuscular, subcutaneous, intravenous, intradermal, intraocular,intratracheal, intracisternal, intraperitoneal, and epidural)administration. Preferably the compositions are administeredintravenously.

The compositions may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association one or morecompositions of the present invention and one or more pharmaceuticalcarriers or excipients.

The triciribine compounds and and one or more taxanes and compositionsthereof can be formulated into suitable pharmaceutical preparations suchas solutions, suspensions, tablets, dispersible tablets, pills,capsules, powders, sustained release formulations or elixirs, for oraladministration or in sterile solutions or suspensions for parenteraladministration, as well as transdermal patch preparation and dry powderinhalers. In one embodiment, the triciribine compounds described aboveare formulated into pharmaceutical compositions using techniques andprocedures well known in the art (see, e.g., Ansel Introduction toPharmaceutical Dosage Forms, Fourth Edition 1985, 126).

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives thereof may be mixed with oneor more suitable pharmaceutical carriers. The compounds of the inventionmay be derivatized as the corresponding salts, esters, enol ethers oresters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids,bases, solvates, hydrates or prodrugs prior to formulation. Theconcentrations of the compounds in the compositions are effective fordelivery of an amount, upon administration, that treats, prevents, orameliorates one or more of the symptoms of the target disease ordisorder. In one embodiment, the compositions are formulated for singledosage administration. To formulate a composition, the weight fractionof compound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

Compositions suitable for oral administration may be presented asdiscrete units such as, but not limited to, tablets, caplets, pills ordragees capsules, or cachets, each containing a predetermined amount ofone or more of the compositions; as a powder or granules; as a solutionor a suspension in an aqueous liquid or a non-aqueous liquid; or as anoil-in-water liquid emulsion or a water-in-oil emulsion or as a bolus,etc.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing a triciribinecompound and optional pharmaceutical adjuvants in a carrier, such as,for example, water, saline, aqueous dextrose, glycerol, glycols,ethanol, and the like, to thereby form a solution or suspension. Ifdesired, the pharmaceutical composition to be administered may alsocontain minor amounts of nontoxic auxiliary substances such as wettingagents, emulsifying agents, solubilizing agents, pH buffering agents,preservatives, flavoring agents, and the like, for example, acetate,sodium citrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents. Methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition, 1975.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having one or more triciribine compounds and oneor more taxanes of the present invention in an inert basis such asgelatin and glycerin, or sucrose and acacia; and mouthwashes, having oneor more of the compositions of the present invention administered in asuitable liquid carrier.

The tablets, pills, capsules, troches and the like can contain one ormore of the following ingredients, or compounds of a similar nature: abinder; a lubricant; a diluent; a glidant; a disintegrating agent; acoloring agent; a sweetening agent; a flavoring agent; a wetting agent;an emetic coating; and a film coating. Examples of binders includemicrocrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone,crospovidones, sucrose and starch paste. Lubricants include talc,starch, magnesium or calcium stearate, lycopodium and stearic acid.Diluents include, for example, lactose, sucrose, starch, kaolin, salt,mannitol and dicalcium phosphate. Glidants include, but are not limitedto, colloidal silicon dioxide. Disintegrating agents includecrosscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Coloring agents include, for example, any of theapproved certified water soluble FD and C dyes, mixtures thereof; andwater insoluble FD and C dyes suspended on alumina hydrate. Sweeteningagents include sucrose, lactose, mannitol and artificial sweeteningagents such as saccharin, and any number of spray dried flavors.Flavoring agents include natural flavors extracted from plants such asfruits and synthetic blends of compounds which produce a pleasantsensation, such as, but not limited to peppermint and methyl salicylate.Wetting agents include propylene glycol monostearate, sorbitanmonooleate, diethylene glycol monolaurate and polyoxyethylene lauralether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

Compositions suitable for topical administration to the skin may bepresented as ointments, creams, gels, and pastes, having one or more ofthe compositions administered in a pharmaceutical acceptable carrier.

Compositions for rectal administration may be presented as a suppositorywith a suitable base including, for example, cocoa butter or asalicylate.

Compositions suitable for nasal administration, when the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken, (i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose). Whenthe carrier is a liquid (for example, a nasal spray or as nasal drops),one or more of the compositions can be admixed in an aqueous or oilysolution, and inhaled or sprayed into the nasal passage.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining one or more of the compositions and appropriate carriers.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats, and solutes which render the formulationisotonic with the blood of the intended recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents andthickening agents. The compositions may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules, andtablets of the kind previously described above.

Pharmaceutical organic or inorganic solid or liquid carrier mediasuitable for enteral or parenteral administration can be used tofabricate the compositions. Gelatin, lactose, starch, magnesiumstearate, talc, vegetable and animal fats and oils, gum, polyalkyleneglycol, water, or other known carriers may all be suitable as carriermedia.

Compositions including triciribine compounds and one or more taxanes maybe used in combination with one or more pharmaceutically acceptablecarrier mediums and/or excipients. As used herein, “pharmaceuticallyacceptable carrier medium” includes any and all carriers, solvents,diluents, or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants, adjuvants, vehicles,delivery systems, disintegrants, absorbents, preservatives, surfactants,colorants, flavorants, or sweeteners and the like, as suited to theparticular dosage form desired.

Additionally, the compositions including triciribine compounds and oneor more taxanes may be combined with pharmaceutically acceptableexcipients, and, optionally, sustained-release matrices, such asbiodegradable polymers, to form therapeutic compositions. A“pharmaceutically acceptable excipient” includes a non-toxic solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type.

It will be understood, however, that the total daily usage of thecompositions will be decided by the attending physician within the scopeof sound medical judgment. The specific therapeutically effective doselevel for any particular host will depend upon a variety of factors,including for example, the disorder being treated and the severity ofthe disorder; activity of the specific composition employed; thespecific composition employed, the age, body weight, general health, sexand diet of the patient; the time of administration; route ofadministration; rate of excretion of the specific compound employed; theduration of the treatment; the triciribine compound and/or the taxaneused in combination or coincidential with the specific compositionemployed; and like factors well known in the medical arts. For example,it is well within the skill of the art to start doses of the compositionat levels lower than those required to achieve the desired therapeuticeffect and to gradually increase the dosage until the desired effect isachieved.

Compositions including triciribine compounds and one or more taxanes arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers to aphysically discrete unit of the composition appropriate for the host tobe treated. Each dosage should contain the quantity of compositioncalculated to produce the desired therapeutic affect either as such, orin association with the selected pharmaceutical carrier medium.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. For example, approximately 1-5 mg per day of acompound disclosed herein can reduce the volume of a solid tumor inmice.

The dosage will depend on host factors such as weight, age, surfacearea, metabolism, tissue distribution, absorption rate and excretionrate. In one embodiment, approximately 0.5 to 7 grams per day of atriciribine compound disclosed herein may be administered to humans.Optionally, approximately 1 to 4 grams per day of the compound can beadministered to humans. In certain embodiments 0.001-5 mg/day isadministered to a human. The therapeutically effective dose level willdepend on many factors as noted above. In addition, it is well withinthe skill of the art to start doses of the composition at relatively lowlevels, and increase the dosage until the desired effect is achieved.

Compositions including triciribine compounds and one or more taxanes maybe used with a sustained-release matrix, which can be made of materials,usually polymers, which are degradable by enzymatic or acid-basedhydrolysis or by dissolution. Once inserted into the body, the matrix isacted upon by enzymes and body fluids. A sustained-release matrix forexample is chosen from biocompatible materials such as liposomes,polylactides (polylactic acid), polyglycolide (polymer of glycolicacid), polylactide co-glycolide (copolymers of lactic acid and glycolicacid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxcylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

The triciribine compounds and one or more taxanes may also beadministered in the form of liposomes. As is known in the art, liposomesare generally derived from phospholipids or other lipid substances.Liposomes are formed by mono- or multi-lamellar hydrated liquid crystalsthat are dispersed in an aqueous medium. Any non-toxic,physiologically-acceptable and metabolizable lipid capable of formingliposomes can be used. The liposome can contain, in addition to one ormore compositions of the present invention, stabilizers, preservatives,excipients, and the like. Examples of lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art.

The triciribine compounds and one or more taxanes may be formulated asaerosols for application, such as by inhalation. These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, in one embodiment, havediameters of less than 50 microns, in one embodiment less than 10microns.

Compositions including the triciribine compounds and one or more taxanesmay be used in combination with other compositions and/or procedures forthe treatment of the conditions described above. For example, a tumormay be treated conventionally with surgery, radiation, or chemotherapycombined with one or more compositions of the present invention and thenone or more compositions of the present invention may be subsequentlyadministered to the patient to extend the dormancy of micrometastasesand to stabilize, inhibit, or reduce the growth of any residual primarytumor.

7.1. Additional Embodiments

The pharmaceutical compositions including triciribine compounds and oneor more taxanes can be formulated according to known methods forpreparing pharmaceutically useful compositions. Formulations aredescribed in a number of sources which are well known and readilyavailable to those skilled in the art. For example, Remington'sPharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack PublishingCompany, 19^(th) ed.) describes formulations which can be used inconnection with the subject invention. Formulations suitable foradministration include, for example, aqueous sterile injectionsolutions, which may contain antioxidants, buffers, bacteriostats, andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and nonaqueous sterile suspensions whichmay include suspending agents and thickening agents. The formulationsmay be presented in unit-dose or multi-dose containers, for examplesealed ampoules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the condition of the sterileliquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

The methods of the present invention, for example, for inhibiting thegrowth of a cancerous cell, can be advantageously combined with at leastone additional therapeutic method, including but not limited tochemotherapy, radiation therapy, therapy that selectively inhibits Rasoncogenic signaling, or any other therapy known to those of skill in theart of the treatment and management of cancer, such as administration ofan anti-cancer agent.

Administration of API-2 (triciribine) as a salt may be carried out.Examples of pharmaceutically acceptable salts are organic acid additionsalts formed with acids which form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, andalpha-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, sulfate, nitrate, bicarbonate, and carbonatesalts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The triciribine compounds and one or more taxanes can be formulated aspharmaceutical compositions and administered to a subject, such as ahuman or veterinary patient, in a variety of forms adapted to the chosenroute of administration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the triciribine compounds and one or more taxanes of the presentinvention may be systemically administered, e.g., orally, in combinationwith a pharmaceutically acceptable vehicle (i.e., carrier) such as aninert diluent or an assimilable edible carrier. They may be enclosed inhard or soft shell gelatin capsules, may be compressed into tablets, ormay be incorporated directly with the food of the patient's diet. Fororal therapeutic administration, the compounds may be combined with oneor more excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Such compositions and preparations should contain at least0.1% of active agent. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of a given unit dosage form. Theamount of the active compound in such therapeutically usefulcompositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the compounds of the invention, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, thecompounds of the invention may be incorporated into sustained-releasepreparations and devices.

The triciribine compounds and one or more taxanes may also beadministered intravenously or intraperitoneally by infusion orinjection. Solutions of the active agents or their salts can be preparedin water, optionally mixed with a nontoxic surfactant. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, triacetin,and mixtures thereof and in oils. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powdersincluding the active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform must be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium including, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating compounds ofthe invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the triciribine compounds and one or moretaxanes may be applied in pure-form, i.e., when they are liquids.However, it will generally be desirable to administer them to the skinas compositions or formulations, in combination with a dermatologicallyacceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds of the invention can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the compounds of the invention to the skinare disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman(U.S. Pat. No. 4,820,508).

Useful dosages of the pharmaceutical compositions of the presentinvention can be determined by comparing their in vitro activity, and invivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

In one non-limiting embodiment, the concentration of the active agent ina liquid composition, such as a lotion, can be from about 0.1-25 wt-%,or from about 0.5-10 wt.-%. In one embodiment, the concentration in asemi-solid or solid composition such as a gel or a powder can be about0.1-5 wt.-%, preferably about 0.5-2.5 wt.-%. In one embodiment, singledosages for injection, infusion or ingestion will generally vary between5-1500 mg, and may be administered, i.e., 1-3 times daily, to yieldlevels of about 0.1-50 mg/kg, for adults. A non-limiting dosage of thepresent invention can be between 7.5 to 45 mg per clay, administeredorally, with appropriate adjustment for the body weight of anindividual.

Accordingly, the present invention includes a pharmaceutical compositionincluding triciribine compounds and one or more taxanes orpharmaceutically acceptable salts thereof, in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor oral, topical or parenteral administration, including an amount oftriciribine compounds and one or more taxanes or a pharmaceuticallyacceptable salt thereof, constitute a preferred embodiment of theinvention. The dose administered to a subject, particularly a human, inthe context of the present invention should be sufficient to affect atherapeutic response in the patient over a reasonable time frame. Oneskilled in the art will recognize that dosage will depend upon a varietyof factors including the condition of the animal, the body weight of theanimal, as well as the severity and stage of the cancer.

A suitable dose is that which will result in a concentration of thetriciribine compounds and one or more taxanes in tumor tissue which isknown to affect the desired response. The preferred dosage is the amountwhich results in maximum inhibition of cancer cell growth, withoutunmanageable side effects. Administration of API-2 (or apharmaceutically acceptable salt thereof) can be continuous or atdistinct intervals, as can be determined by a person of ordinary skillin the art.

Mammalian species which benefit from the disclosed methods for theinhibition of cancer cell growth, include, but are not limited to,primates, such as apes, chimpanzees, orangutans, humans, monkeys;domesticated animals (e.g., pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. Theterms “patient” and “subject” are used herein interchangeably and areintended to include such human and non-human mammalian species.Likewise, in vitro methods of the present invention can be earned out oncells of such mammalian species.

Patients in need of treatment using the methods of the present inventioncan be identified using standard techniques known to those in themedical profession.

The following examples are offered by way of illustration and not by wayof limitation.

8. EXAMPLES 8.1. Example 1 In Vitro Screening

Cell Lines and NCI Diversity Set.

All cell lines can be purchased from ATCC or described previously(Cheng, J. Q., et al. Oncogene, 14: 2793-2801, 1997, West, K. A., et al.Drug Resist. Updat., 5: 234-248, 2002, Satyamoorthy, K., et al. CancerRes. 61: 7318-7324, 2001). The NCI Structural Diversity Set is a libraryof 1,992 compounds selected from the approximately 140,000-compound NCIdrug depository. In-depth data on the selection, structures, andactivities of these diversity set compounds can be found on the NCIDevelopmental Therapeutics Program web site.

Screening for Inhibition of Akt-transformed Cell Growth. AKT2Transformed NIH3T3 Cells or LXSN Vector-Transfected NIH3T3 Control Cells(Cheng, J. Q., et al. Oncogene, 14: 2793-2801, 1997) are plated into96-well tissue culture plate. Following treatment with 5 M of NCIDiversity Set compound, cell growth can be detected with CellTier 96 OneSolution Cell Proliferation kit (Promega). Compounds that inhibit growthin AKT2-transformed but not LXSN-transfected NIH3T3 cells are consideredas candidates of Akt inhibitor and subjected to further analysis.

In Vitro Protein Kinase, Cell Survival and Apoptosis Assays.

In vitro kinase is performed as previously described (see, for example,Jiang, K., Coppola, et al. Mol. Cell. Biol., 20:139-148, 2000). Cellsurvival is assayed with MTS (Promega). Apoptosis was detected withannexin V, which is performed as previously described (Jiang, K.,Coppola, et al. Mol. Cell. Biol., 20:139-148, 2000). Recombinant Akt andPDK1 are purchased from Upstate Biotechnology Inc.

Results

Identification of Small Molecule Inhibitor of Akt Signaling Pathway,API-2.

Frequent alterations of Akt has been detected in human cancer anddisruption of Akt pathway induces apoptosis and inhibits tumor growth(Jetzt, A., et al. Cancer Res., 63: 697-706, 2003). Thus, Akt isconsidered as an attractive molecular target for development of novelcancer therapeutics. To identify small molecule inhibitor(s) of Akt, achemical library of 1,992-compounds from the NCI (the NCI Diversity Set)is evaluated for agents capable of inhibition of growth inAKT2-transformed but not empty vector LXSN-transfected NIH3T3 cells.Repeated experiments showed that 32 compounds inhibited growth only inAKT2-transformed cells. The most potent of these compounds, API-2 (NCIidentifier: NSC 154020), can suppress cell growth at a concentration of50 nM. FIG. 1A shows the chemical structure of API-2, which is alsoknown as triciribine (Schweinsberg, P. D., et al. Biochem Pharmacol.,30: 2521-2526, 1981). The fact that API-2 inhibits selectively AKT-2transformed cells over untransformed parental cells prompted us todetermine whether API-2 is an inhibitor of AKT2 kinase. To this end,AKT2 is immunoprecipitated with anti-AKT2 antibody from AKT-2transformed NIH3T3 cells following treatment with API-2. AKT2immunoprecipitates were immunoblotted with anti-phospho-Akt antibodies.As shown in FIG. 1B, API-2 significantly inhibited AKT2 phosphorylationat both threonine-309 and serine-474, which are required for fullactivation of AKT2 (Datta, S. R., et al. Genes Dev. 13: 2905-2927,1999). As three isoforms of Akt share high homology and similarstructure, the effect of API-2 on their kinase activities is evaluated.HEK293 cells are transfected with HA-Akt1, -AKT2 and -AKT3,serum-starved overnight and treated with API-2 for 60 min prior to EGF(50 ng/ml) stimulation. Triple experiments showed that API-2 suppressedEGF-induced kinase activity and phosphorylation of Akt1, AKT2 and AKT3(FIG. 1C). However, kinase activity of recombinant constitutively activeAKT2 (Myr-AKT2) is not inhibited by API-2 in an in vitro kinase reaction(FIG. 1D), suggesting that API-2 does not directly inhibit Akt in vitroand that API-2 neither functions as ATP competitor nor as the substratecompetitor that binds to active site of Akt.

API-2 does not Inhibit Known Upstream Activators of Akt.

It has been well documented that Akt is activated by extracellularstimuli and intracellular signal molecules, such as active Ras and Src,through a PI3K-dependent manner. Therefore, API-2 inhibition of Aktcould result from targeting upstream molecule(s) of Akt. As PI3K andPDK1 are direct upstream regulators of Akt (Datta, S. R., et al. GenesDev. 13: 2905-2927, 1999), whether API-2 inhibits PI3K and/or PDK1 isexamined. HEK293 cells are serum-starved and then can be treated withAPI-2 or PI3K inhibitor, wortmannin, for 30 min prior to EGFstimulation. PI3K is immunoprecipitated with anti-p110 antibody. Theimmunoprecipitates are subjected to in vitro PI3K kinase assay usingPI-4-P as a substrate. As shown in FIG. 2A, the EGF-induced PI3Kactivity is inhibited by wortmannin but not by API-2. To evaluate theeffect of API-2 on PDK1, an assay in which recombinant PDK1 promotes thethreonine-309 phosphorylation of AKT2 peptides is used in the presenceof lipid vesicles containing phosphotidylinositol. As shown in FIG. 2B,the assay is potently inhibited by the control PDK1 inhibitorstaurosporine (IC50=5 nM). In contrast, API-2 displayed only 21%inhibition of the assay at the highest concentration tested (5.1 M). Tofurther evaluate the effect of API-2 on PDK1 activation, theautophosphorylation level of PDK1 at serine-241, a residue that isphosphorylated by itself and is critical for its activity is examined(Datta, S. R., et al. Genes Dev. 13: 2905-2927, 1999), following API-2treatment of HEK293 cells. Triplicate experiments show thatphosphorylation levels of PDK1 are not inhibited by API-2 (FIG. 2B).However, PI3K inhibitor wortmannin can inhibit EGF-stimulated PDK1 (FIG.2B).

API-2 is Highly Selective for the Akt Over PKC, PKA, SGK, STAT, JNK,p38, and ERK Signaling Pathways.

Akt belongs to AGC (PKA/PKG/PKC) kinase family, which also include PKA,PKC, serum- and glucocorticoid-inducible kinase (SGK), p90 ribosomal S6kinase, p70^(S6K), mitogen- and stress-activated protein kinase andPKC-related kinase. Among AGC kinase family, protein structures of PKA,PKC and SGK are more close to Akt kinase than other members. Therefore,next examined are the effects of API-2 on the enzymatic activities ofthese 3 kinases. HEK293 cells are transfected with HA-tagged PKA, PKC orSGK. In vitro kinase assay and immunoblotting analysis show that thekinase activities of PKA and PKCα are inhibited by PKAI and Ro 31-8220,a PKC inhibitor, respectively, whereas API-2 exhibits no effect on theiractivities (FIGS. 2C and 2E). Further, serum-induced SGK kinase activityis attenuated by wortmannin but not by API-2 (FIG. 2D). In addition, itis determined whether API-2 has effect on other oncogenic survivalpathways. Western blotting analyses with commercially availableanti-phospho-antibodies reveals that phosphorylation levels of Stat3,JNK, p38 and Erk1/2 were not affected by API-2 treatment (FIG. 2F).These data indicate that API-2 specifically inhibits Akt signalingpathway.

API-2 Suppresses Cell Growth and Induces Apoptosis inAkt-Overexpressing/Activating Human Cancer Cell Lines.

The ability of API-2 to selectively inhibit the Akt pathway suggeststhat it should inhibit proliferation and/or induces apoptosispreferentially in those tumor cells with aberrant expression/activationof Akt. As activation of Akt in human malignancies commonly results fromoverexpression of Akt or PTEN mutations, API-2 is used to treat thecells that express constitutively active Akt, caused by overexpressionof AKT2 (OVCAR3, OVCAR8, PANC1 and AKT2-transformed NIH3T3) or mutationsof the PTEN gene (PC-3, LNCaP, MDA-MB-468), and cells that do not(OVCAR5, DU-145, T47D, COLO357 and LXSN-NIH3T3) as well as melanomacells that are activated by IGF-1 to activate Akt or do not respond togrowth stimulation by IGF-1 (Satyamoorthy, K., et al. Cancer Res. 61:7318-7324, 2001). Immunoblotting analysis showed that phosphorylationlevels of Akt are inhibited by API-2 only in the cells expressingelevated Akt or responding to IGF-1 simulation (FIG. 3A). Accordingly,API-2 inhibited cell growth to a much higher degree inAkt-overexpressing/activating cells as compared to those with low levelsof Akt. As shown in FIG. 3B, API-2 treatment inhibited cellproliferation by approximate 50-60% in Akt-overexpressing/activatingcell lines, LNCaP, PC-3, OVCAR3, OVCA8, PANC1, MDA-MB-468, and WM35,whereas only by about 10-20% in DU145, OVCAR5, COLO357, T47D and WM852cells, which exhibit low levels of Akt or do not respond to growthstimulation by IGF-1. Moreover, API-2 induces apoptosis by 8-fold(OVCAR3), 6-fold (OVCAR8), 6-fold (PANC1), and 3-fold (AKT2-NIH3T3). Nosignificant difference of apoptosis is observed between API-2 andvehicle (DMSO) treatment in OVCAR5, COLO357 and LXSN-NIH3T3 cells. Thus,API-2 inhibits cell growth and induces apoptosis preferentially in cellsthat express aberrant Akt.

API-2 in Combination with Taxol Acts Synergistically to Suppress CellGrowth and Induce Apoptosis in Akt-Overexpressing/Activating HumanCancer Cell Lines.

The ability of API-2 and Taxol to selectively inhibit the Akt pathwaysuggests that it should inhibit proliferation and/or induces apoptosispreferentially in those tumor cells with aberrant expression/activationof Akt. As activation of Akt in human malignancies commonly results fromoverexpression of Akt or PTEN mutations, API-2 and Taxol in comvinationare used to treat the cells that express constitutively active Akt,caused by overexpression of AKT2 (OVCAR3, OVCAR8, PANC1 andAKT2-transformed NIH3T3) or mutations of the PTEN gene (PC-3, LNCaP,MDA-MB-468), and cells that do not (OVCAR5, DU-145, T47D, COLO357 andLXSN-NIH3T3) as well as melanoma cells that are activated by IGF-1 toactivate Akt or do not respond to growth stimulation by IGF-1. (FIG.3A). API-2 and Taxol inhibits cell growth to a much higher degree inAkt-overexpressing/activating cells as compared to those with low levelsof Akt. As shown in FIG. 9, API-2/Taxol treatment inhibits cellproliferation by approximate 50-65% in Akt-overexpressing/activatingcell lines, LNCaP, PC-3, OVCAR3, OVCA8, PANC1, MDA-MB-468, and WM35,whereas only by about 10-20% in DU145, OVCAR5, COLO357, T47D and WM852cells, which exhibit low levels of Akt or do not respond to growthstimulation by IGF-1. Thus, API-2 and Taxol acts synergistically toinhibit cell growth and induce apoptosis preferentially in cells thatexpress aberrant Akt.

API-2 Inhibits Downstream Targets of Akt.

It has been shown that Akt exerts its cellular effects throughphosphorylation of a number of proteins (Datta, S. R., et al. Genes Dev.13: 2905-2927, 1999). More than 20 proteins have been identified as Aktsubstrates, including the members of Forkhead protein family (FKHR, AFXand FKHRL1), tuberlin/TSC2, p70^(S6K), GSK-3β, p21^(WAF1/Cip1),p27^(kip1), MDM2, Bad, ASK1 and IKKα etc. It is next examined whetherAPI-2 inhibits downstream targets of Akt. As anti-phospho-tuberlin,-Bad, -AFX, and -GSK-3 antibodies are commercially available, therefore,the effect of API-2 on their phosphorylation induced by Akt wasdetermined. Following API-2 (1 μM) treatment, OVCAR3 cells was lysed andimmunoblotted with the individual anti-phospho-antibody. FIG. 4A showsthat API-2 considerably inhibited the phosphorylation levels of tuberlinleading to stabilization and upregulation of tuberin (Dan, H. C., et al.J. Biol. Chem., 277: 35364-35370, 2002). The phosphorylation levels ofBad, GSK-3β, and AFX are partially attenuated by API-2. These datasuggest that API-2 induces cell death and cell growth arrest byinhibiting phosphorylation of its downstream targets. API-2 inhibitionof Akt downstream targets at different degrees could be due to the factthat phosphorylation sites of these targets are also regulated by otherkinase(s), for instance, Bad serine-136 is phosphorylated by PAK1 inaddition to Akt (Schurmann, A., et al. Mol. Cell. Biol., 20: 453-461,2000).

EXAMPLE 2 Antitumor Activity in the Nude Mouse Tumor Xenograft Model

Tumor cells can be harvested, suspended in PBS, and can be injected s.c.into the right and left flanks (2×10⁶ cells/flank) of 8-week-old femalenude mice as reported previously (Sun, J., Blaskovic, et al. CancerRes., 59: 4919-4926, 1999). When tumors reach about 100-150 mm³, animalsare randomized and dosed i.p. with 0.2 ml vehicle of the triciribinecompound and/or the taxane daily. Control animals receive DMSO (20%)vehicle, whereas treated animals can be injected with API-2 (1mg/kg/day) in 20% DMSO.

API-2 Inhibits the Growth of Tumors in Nude Mice that Overexpress Akt.

Frequent overexpression/activation and/or amplification of AKT1 and AKT2in human ovarian and pancreatic cancer was shown (Cheng, J. Q., andNicosia, S. V. AKT signal transduction pathway in oncogenesis. In SchwabD, Editor, Encyclopedic Reference of Cancer. Berlin Heidelberg and NewYork: Springer; 2001. pp 35-7). Inhibition of Akt pathway by inhibitorsof PI3K, HSP70, Src and farnesyltransferase resulted in cell growtharrest and induction of apoptosis (Solit, D. B., et al. Cancer Res., 63:2139-2144, 2003, Xu, W., et al. Cancer Res., 63: 7777-7784, 2003). Arecent study showed that the tumor growth of xenografts with elevatedAkt was also significantly inhibited by intratumoral injection ofadenovirus of dominant negative Akt (Jetzt, A., et al. Cancer Res., 63:697-706, 2003). Because API-2 inhibits Akt signaling and inducesapoptosis and cell growth arrest only in cancer cells with elevatedlevels of Akt (FIG. 3), the growth of tumors with elevated levels of Aktshould be more sensitive to API-2 than that of tumors with low levels ofAkt in nude mice. To this end, s.c. Akt-overexpressing cells (OVCAR3,OVCAR8 and PANC-1) are s.c. implanted into the right flank, and thosecell lines that express low levels of Akt (OVCAR5 and COLO357) into theleft flank of mice. When the tumors reach an average size of about100-150 mm³, the animals are randomized and treated i.p. with eithervehicle or API-2 (1 mg/kg/day). As illustrated in FIG. 4B, OVCAR-5 andCOLO357 tumors treated with vehicle grew to about 800-1,000 mm³ 49 daysafter tumor implantation. OVCAR3, OVCAR8 and PANC1 tumors treated withvehicle control grew to about 700-900 mm³ 49 days after tumorimplantation. API-2 inhibited OVCAR3, OVCAR8 and PANC1 tumor growth by90%, 88% and 80%, respectively. In contrast, API-2 has little effect onthe growth of OVCAR5 and COLO357 cells in nude mice (FIGS. 4B-4D anddata not shown). At dose 1 mg/kg/day, API-2 had no effects on bloodglucose level, body weight, activity and food intake of mice. In treatedtumor samples, Akt activity was inhibited by API-2 without change oftotal Akt content (FIG. 4E). Taken together, these results indicate thatAPI-2 selectively inhibits the growth of tumors with elevated levels ofAkt.

EXAMPLE 3 TCN Directly Inhibits Wild Type Akt Kinase Activity

API-2 (TCN) can directly inhibit wild type Akt kinase activity inducedby PDK1 in vitro (FIG. 1). This result supports that API-2 is a directAkt inhibitor and that the underlying mechanism may be API-2 binding toPH domain and/or threonine-308 of Akt. An in vitro kinase assay isperformed with recombinant of PDK1 and Akt in a kinase buffer containingphosphatidylinositol-3,4,5-P3 (PIP3), API-2 and histone H2B assubstrate. After incubation of 30 min, the reactions were separated bySDS-PAGE and exposed in a film.

EXAMPLE 4 TCN is Effective in Cancer Resistant Cells

The effects of TCN (API-2) are tested in cisplatin, paclitaxel, andtamoxifen resistant A270CP, C-13, OVCAR433 and MCF7/TAM cells. API-2overcame cisplatin, paclitaxel, and tamoxifen resistance in these cells

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

1-27. (canceled)
 28. A method for treating a subject having a tumor orcancer, which tumor or cancer overexpresses AKT kinase comprising: (i)determining whether the subject has a tumor or cancer that overexpressesAKT kinase; (ii) if said subject has a tumor or cancer thatoverexpresses AKT kinase, a. administering to said subject: i. at leastone compound of formula I selected from the group consisting of thefollowing compounds:

 wherein each R₂′, R₃′, and R₅′ is independently hydrogen; optionallysubstituted phosphate or phosphonate; mono-, di-, or triphosphate; acyl;lower acyl; alkyl; lower alkyl; amide; sulfonate ester; alkyl sulfonateester; arylalkyl sulfonate ester; sulfonyl; methanesulfonyl; benzylsulfonyl, wherein the phenyl group of said benzyl is optionallysubstituted with one or more halo, hydroxyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate; optionally substitutedarylsulfonyl; a lipid; phospholipid; an amino acid; a carbohydrate; apeptide; or cholesterol; or other pharmaceutically acceptable leavinggroup that, in vivo, provides a compound of said formula I wherein R₂′,R₃′ or R₅′ is independently H or mono-, di- or tri-phosphate;  whereinR^(x) and R^(y) are independently hydrogen; optionally substitutedphosphate; acyl; lower acyl; amide; alkyl; lower alkyl; aromatic;polyoxyalkylene; polyethyleneglycol; optionally substitutedarylsulfonyl; a lipid; a phospholipid; an amino acid; a carbohydrate; apeptide; or cholesterol; or other pharmaceutically acceptable leavinggroup; and  wherein R₁ and R₂ each are independently H, optionallysubstituted straight chained, branched or cyclic alkyl, lower alkyl,alkenyl, or alkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl orheteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonyl, alkylsulfonyl, arylsulfonyl, or aralkylsulfonyl; ii. acompound of formula II or a salt thereof:

 wherein  R¹⁰ and R¹¹ each are independently H, optionally substitutedstraight chained, branched or cyclic alkyl (including lower alkyl),alkenyl, or alkynyl, aryl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl orheteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl,  wherein atleast 0.1 mg of the compound of formula II is administered to saidsubject; and iii. a pharmaceutically acceptable carrier.
 29. The methodof claim 28, wherein the compound of formula I is triciribine.
 30. Themethod of claim 28, wherein the compound of formula I is triciribinephosphate.
 31. The method of claim 28, wherein the compound of formula Iis triciribine phosphonate.
 32. The method of claim 28, wherein thecompound of formula I is present in a dose amount of at least 2 mg/m².33. The method of claim 28, wherein the compound of formula I is presentin an amount of less than 15 mg/m².
 34. The method of claim 28, whereinthe administration is parenteral administration.
 35. The method of claim34, wherein the parenteral administration is intravenous administration.36. The method of claim 28, wherein the administration is oraladministration.
 37. The method of claim 28, suitable for intravenousadministration.
 38. The method of claim 28, wherein the compound offormula II or salt thereof is present in a dose of at least 10 mg. 39.The method of claim 28, wherein the compound of formula II or saltthereof is present in a dose of at least 50 mg.
 40. The method of claim28, wherein the tumor or cancer is pancreatic, prostate, colo-rectaland/or ovarian.
 41. The method of claim 28, wherein the administrationof a compound of formula I and the compound of formula II or saltthereof is concurrently administered.
 42. The method of claim 28,wherein the administration of a compound of formula I is followed by theadministration of the compound of formula II or salt thereof.
 43. Themethod of claim 28, wherein the administration of the compound offormula II or salt thereof is followed by the administration of acompound of formula I.